Delivery of biomolecules to immune cells

ABSTRACT

A method and device for preferentially delivering a compound such as an antigen to the cytosol of an immune cell. The method comprises passing a cell suspension comprising the target immune cell through a microfluidic device and contacting the suspension with the compound(s) or payload to be delivered.

RELATED APPLICATIONS

This application claims the benefit of priority to PCT Application No.PCT/US2015/058489, filed Oct. 30, 2015, which claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.62/073,548, filed Oct. 31, 2014, each of which are incorporated hereinby reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.GM101420, AI112521, AI111595, and AI069259 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to the delivery of materials to cells.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“38172-508001WO_Sequence_Listing.txt,” which is 2.31 kilobytes in size,and which was created Oct. 30, 2015 in the IBM-PC machine format, havingan operating system compatibility with MS-Windows, which is contained inthe text file filed Oct. 30, 2015 as part of this application.

BACKGROUND OF THE INVENTION

Delivery of macromolecules, such as polysaccharides, proteins, ornucleic acids, to the cell cytoplasm can transiently or permanentlyalter cell function for research or therapeutic purposes. However,existing techniques for intracellular delivery to primary immune cells,especially resting lymphocytes, have limitations. Electroporationresults in considerable cellular toxicity. Viral vectors are unable toinfect resting lymphocytes. Cell membrane penetrating (or transduction)peptides do not efficiently transfect primary lymphocytes. Antibody-drugcomplexes and conjugates require specific antibodies for each cell typeand distinct designs to carry different payloads. Furthermore, they areexpensive to produce and potentially immunogenic. Aptamer-siRNA chimericRNAs have been shown to cause targeted gene knockdown in vivo, withoutany toxicity or immune activation, but have only been used to deliversmall RNAs and they require identifying specific targeting aptamers foreach cell of interest. Advances in nanoparticle and liposome basedtechnologies have resulted in improved intracellular delivery of drugsand antigens to phagocytic antigen presenting cells, such as dendriticcells and monocyte/macrophages, but are ineffective for lymphocytes.Most of these methods lead to endosomal uptake of the payload, and onlya very small proportion of the payload (estimated as ˜1-2%) escapes fromthe endosome to the cytosol, where it needs to traffic for biologicalactivity. Many of these techniques also result in accumulation ofnonbiodegradable packaging or delivery material in the cell, which mayaffect cell function. Thus there is a need for alternative techniquescapable of efficient and nontoxic delivery of a variety ofmacromolecules to immune cells.

SUMMARY OF THE INVENTION

The invention provides a solution to previous problems associated withdelivery of compounds or compositions to immune cells. Prior to theinvention, introduction of compounds, e.g., proteins, nucleic acids,carbohydrates, was difficult and occurred inefficiently and/or requiredthe presence of undesirable mediators such as toxic compounds or viralvectors. According to the invention, a method for engineering of immunecell function comprises intracellular delivery of compounds bytransiently disrupting a membrane surrounding the cytoplasm of theimmune cell. For example, a viral vector-free method for preferentiallydelivering a compound to the cytosol of an immune cell includes a stepof passing a cell suspension comprising the target immune cell through amicrofluidic device and contacting the suspension with the compound(s)or payload to be delivered. The device comprises a constriction lengthof 10-60 μm and a constriction width of 3-8 μm, e.g., 3-4 μm or 4 μm.

For example, the device comprises a constriction of a diameter of 2μm-10 μm. In preferred embodiments relating to naïve T and B cells, thedevice comprises a constriction having a length of about 10, 15, 20, 25,30, or 10-30 μm, a width of about 3, 3.5, 4, or 3-4 μm, a depth of about15, 20, 25, or 15-25 μm, and/or an about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 5-15 degree angle.

Following passage through the constriction, the amount of compounddelivered to an immune cell is at least 10% greater (e.g., 20%, 50%,2-fold, 5-fold, 10-fold, or greater) than that delivered to a non-immunecell or compared to the amount delivered to an immune cell in theabsence of cell squeezing, e.g., by endocytosis alone. For example, theimmune cell comprises a B cell, T cell, macrophage, or dendritic cell.For delivery of payload preferentially to immune cells, an exemplarydevice is characterized by one or more channels comprising aconstriction length of 30 μm and a constriction width of 4 μm throughwhich the cells pass.

A temperature of 0 to 45 degrees Celsius is used during cell treatment,e.g., 0-25° C. For example, treatment of naïve T cells, B cells and/ormonocytes is carried out at temperature of 4-8° C., e.g., on ice. Inanother example, dendritic cells, activated T cells, and/or activated Bcells are treated using the device at temperatures of 20-25° C., e.g.,at typical ambient room temperature.

The payload contains any molecule or compound sought to be delivered tothe cytoplasm of an immune cell. For example, the compound comprises anantigen, e.g., a disease-associated antigen such as a tumor antigen,viral antigen, bacterial antigen, or fungal antigen. The antigen may bepurified or in a mixture of other components, e.g., the antigen ispresent in a cell lysate such as a tumor cell lysate or lysate ofbiopsied infected or disease-affected tissue from a subject, e.g., asubject suffering from an infectious disease. In some example, theantigen comprises a whole, full-length (or un-processed) proteinantigen, e.g., a protein or peptide that exceeds a length of 7, 8, 9, or10 amino acids. Other cargo molecules include nucleic acids such assiRNA, mRNA, miRNA, coding or non-coding oligonucleotides as well assmall molecules, e.g., small molecule probes. Nucleic acids such as DNA,e.g., expression vectors such as plasmids, are also delivered in thismanner without the need for a viral vector.

In some examples, the immune cell is in a resting state compared to anactivated state. For example, cells are characterized by expression ofthe following markers: CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4,CD28, CD3, CD62L, CCR7, CX3CR1 and CXCR5, each of which may bemanipulated (increased or reduced by introducing molecules into theimmune cells using the methods described). Cell suspensions includeprocessed cells, e.g., resuspended buffy coat cells (fractionated whiteblood cells) or whole blood. Naïve immune cells, e.g., T cells, arecharacterized by comparatively low levels of expression of CD25, CD80,CD86, PD-1, and CTLA-4 and by comparatively high level of CCR7 (comparedto activated cells).

The device for preferentially delivering a compound to an immune cellcompared to a non-immune cell, comprises at least one microfluidicchannel, e.g., in the form of a syringe, or a plurality of channels,e.g., in the form of a microchip or microfluidic device. For example,the channel comprises a constriction length of 30 μm and a constrictionwidth of 4 μm.

The invention also includes a method for engineering of immune cellfunction by intracellular delivery of compounds, which delivery ismediated by transiently disrupting a membrane surrounding the cytoplasmof the immune cell and delivering into the cytosol an antigen. Forexample, the antigen comprises a length of greater than 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acids and wherein the immune cell processes theantigen into a peptide that is less than 11 amino acids in length. Thecell then displays the shorter, processed peptide, a class Ihistocompatibility antigen restricted processed form of the antigen, ona surface of the immune cell. For example, peptides for MHC/HLAprocessed for class I presentation to CD8+ T cells or cytotoxic T cellsare in the range of 8-10 residues, and peptides processed for MHC/HLAclass II presentation to CD4+ T cells or helper cells are in the rangeof 14-20 residues. Peptides shorter than 8 residues (e.g., 2, 3, 4, 5,6, or 7 residues) are useful for presentation to other T cell types orNK cells.

For example, the cell membrane is disrupted by passing the immune cellthrough a constriction of a diameter of 2 μm-10 μm. The antigendelivered by cell squeezing into the cytosol is a full-length,unprocessed protein or a peptide that must be processed to a size/lengthsuitable for binding to a histocompatibility antigen for antigenpresentation by the antigen presenting cell. The method of engineeringimmune cell function may further comprise contacting the antigen-loadedimmune cell with an effector T cell and activating a cytotoxic T cellimmune response. In some examples, squeezed antigen-loaded immune cellcomprises a B cell, dendritic cell or macrophage. In other examples, thesqueezed antigen-loaded immune cell comprises a T cell. In either case,the squeezed antigen loaded immune cell comprises at least 10%, 25%,50%, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, or more antigenor other payload composition compared to an immune cell contacted withthe same antigen or payload in the absence of squeezing, e.g., uptake byendocytosis or pinocytosis alone.

The function, activity, or activation state of the immune cell isaltered following such treatment. For example, a method for conferringan antigen presenting phenotype on a T cell is carried out by deliveringan antigen, e.g., a whole, unprocessed protein or fragment thereof, tothe cytosol of a T cell by passing the T cell through a microfluidicdevice as described above. For example, the device comprises aconstriction of a diameter of about 2 μm-10 μm and the T cell comprisesa class I histocompatibility antigen-restricted, processed form of theantigen on a surface of the T cell following passage through themicrofluidic device. The method may further comprise contacting thefirst (squeezed,antigen-loaded) T cell with a second T cell, the secondT cell comprising a class I histocompatibility antigen restrictedcytotoxic T cell phenotype. Exemplary antigens include one or more tumorantigens, e.g., a mixture of tumor antigens such as a tumor biopsylysate, or a viral antigen. Production of antigen-loaded T cells in thismanner are used in vitro and/or in vivo to elicit a cytotoxic T cellresponse. Thus, the use of cell-squeezed, antigen loaded T cell toactivate a cytotoxic T cell response specific for the antigen isencompassed in the invention. For example, such T cells confer clinicalbenefit by killing tumor cells and/or killing virally-infected cells,depending upon the antigen delivered/loaded.

The compositions described herein are purified. Purified compounds areat least 60% by weight (dry weight) the compound of interest.Preferably, the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by weight the compound ofinterest. Purity is measured by any appropriate standard method, forexample, by column chromatography, polyacrylamide gel electrophoresis,or HPLC analysis. For example, the compound, e.g., protein antigen, hasbeen separated from one or more compounds with which is occurs innature. In the case of cells, a purified population is at least 75%,85%, 90%, 95%, 98%, 99% or 100% the cell type of choice. Methods orpurifying or enriching for a particular cell type are well known,including segregating by size or cell surface marker expression usingdevice such as cell sorting machines.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a series of diagrams showing a delivery system. To set up thesystem, each microfluidic chip is mounted into a holder that allows itto interface with polycarbonate fluid reservoirs. To operate the system,the macromolecules to be delivered are mixed with the cells, loaded intothe device reservoir in a volume of about 30-15 μl and then connected tothe pressure source to induce flow through the microfluidic channels.FIG. 1B is a diagram showing cell deformation and payload delivery. Asthe cells flow through the channels, they deform at the constrictions,resulting in membrane disruption. Macromolecules in the fluid thendiffuse through the disrupted membrane and remain trapped in the cellafter its membrane is repaired. These figures demonstrate delivery bycell squeezing.

FIGS. 2A and B are a histogram and a bar graph showing dextran andantibody delivery to murine immune cells. FIG. 2A shows representativehistograms of T cells, B cells and myeloid cells (CD11 b⁺) treated bythe CellSqueeze device to deliver APC-labeled IgG1. FIG. 2B showsdelivery efficiency. All results were measured by flow cytometry withinan hour of treatment. Dead cells were excluded by propidium iodidestaining. Data in FIG. 2B (mean±SD) are from 3 independent experiments.Untreated cells were not put through the device or exposed to thebiomolecules. The ‘no device’ samples were incubated with thebiomolecules, but were not treated by the device. This control is meantto account for surface binding, endocytosis and other backgroundeffects.

FIGS. 3A-D are graphs showing delivery of dextrans, antibodies and siRNAto human immune cells. In FIG. 3A, human T cells and MDDCs were testedfor delivery of cascade blue labeled 3 kDa dextran, fluorescein labeled70 kDa dextran, and APC labeled IgG1. The representative histograms fora 30-4 (T cells) and 10-7 (MDDCs) device (left) and replicates acrossdevice designs (right) are displayed. FIG. 3B shows SiRNA mediatedknockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCsrespectively. Different siRNA concentrations and device designs weretested to assess knockdown dependence on dose or constriction size. FIG.3C shows that human regulatory T cells also showed significant knockdownof CD4 expression in response to treatment by a 30-4 device. Dead cellswere excluded for delivery or knockdown analysis. FIG. 3D shows acomparison of device performance in T cells to nucleofection by Amaxa.Protein expression 72 hrs after delivery of siRNA against CD4 is shownfor the two systems. Cell viability after treatment by the two methodsis also shown.

FIGS. 4A-B are graphs showing inhibition of HIV infection by targetedknockdown of endogenous and viral genes. In Figure A, intracellularstaining for the p24 antigen was used as an indicator of HIV infectionlevel in treated human CD4⁺ T cells 24 hrs after infection. In thesestudies, vif and/or gag, siRNA was delivered 24 hrs prior to infectionwhile CD4 siRNA was delivered 48 hrs prior to infection. FIG. 4N showsmedian fluorescence intensity of the p24 antigen stain across repeats(min. N=4) of the experimental conditions. Data is represented as mean+1standard error.

FIG. 5 is a diagram of a vaccination method.

FIG. 6 is a series of line graphs showing uptake of 3 kDa and 70 kDadextran and antibody to murine primary immune cells. The gating used tocalculate delivery efficiency values is shown. These data correspond toexperiments presented in FIG. 2. Grey histograms represent untreatedcells, black represents cells that were exposed to the materials but nottreated by the device, red represents cells that were treated by thedevice in the presence of the target biomolecules.

FIG. 7 is a series of bar graphs showing cell viability datacorresponding to the experiments presented in FIG. 2. *** indicatedp<0.001 when comparing viability of cells treated with 30-4 device to nodevice or untreated cases. Changes in viability of B cells and myeloidcells treated with the device were not significantly different from theuntreated or no device cases.

FIG. 8 is a series of graphs showing delivery of dextran and antibodiesto bone marrow-derived dendritic cells (BMDCs). BMDCs were generatedfrom C57BL6 mice by culturing bone marrow cells in GM-CSF containingmedia for 8 days. Cascade blue-labeled 3 kDa dextran,fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1 were deliveredusing two device designs, 10-6 and 30-6.

FIG. 9 is a graph showing correlation of antibody and dextran delivery.Dextran (3 kDa and 70 kDa) and antibody delivery to T cells using the30-4 device (grey dots; center, upper right) compared to incubation withthe material, i.e. no device (black dots; lower left).

FIG. 10 is a bar graph showing viability of human CD4+ T cells. Cellsthat pass through the device have reduced viability when compared tountreated controls, but have better viability than cells that haveundergone nucleofection. One-way ANOVA followed by Boneferroni's testwas used to calculate statistical significance. * indicates p<0.05 and*** indicates p<0.001. Other groups of comparison did not showsignificantly different viability (i.e. 10-4 compared to untreated or30-4, and 30-4 compared to nucleofection).

FIG. 11 is a series of bar graphs showing delivery (top) and viability(bottom) Results from testing different device designs for human MDDCs.Cascade blue labeled 3 kDa dextran, fluorescein labeled 70 kDa dextran,and APC labeled IgG1 isotype control antibodies were delivered using 6different device designs and using Amaxa nucleofection. Viability anddelivery results were measured immediately after treatment.

FIG. 12 is a series of graphs showing Alexa 488 or Alexa 647 labeledsiRNA and 3 kDa cascade blue labeled dextran were deliveredsimultaneously to human CD4 T cells by a 10-4i device and murine B cellsby a 30-5×5i device. The data indicate that delivery of the twomaterials correlates closely. This result is consistent with theproposed diffusive delivery mechanism, i.e. delivery efficacy is mostlydependent on material size rather than chemical structure.

FIG. 13 is a graph showing CD45RA expression. siRNA against CD45RA wasdelivered to human T cells by a 10-4 device. Knockdown was measured byflow cytometry 72 hours post-treatment.

FIG. 14 is a bar graph showing CD4 mRNA knockdown (as measured by PCR 48hours after delivery).

FIG. 15 is a series of bar graphs showing expression levels of CD4 inCD4+ human T cells over 2 weeks post-treatment as measured by flowcytometry. CD3 levels were also measured as a control gene.

FIG. 16 is a series of graphs showing delivery of model cargo, dextran,to human monocytes. Monocytes were derived from human blood. Cascadeblue labeled 3 kDa dextran, and fluorescein labeled 70 kDa dextran weredelivered using four different device designs at two different operatingpressures. The Opsi case corresponds to controls that were only exposedto dextran but not treated by the device. Viability was measured bypropidium iodide staining.

FIG. 17 is a series of graphs showing delivery of dextran to human Bcells. B cells were derived from human blood. Cascade blue labeled 3 kDadextran, and fluorescein labeled 2 MDa dextran were delivered using fivedifferent device designs at two different operating pressures. The Opsicase corresponds to controls that were only exposed to dextran but nottreated by the device. Viability was measured by propidium iodidestaining.

FIG. 18 is a bar graph showing measured protein levels of DC-Sign 72 hrsafter treatment. Protein knockdown was measured across 6 differentdevice designs and compared to nucleofection. Note that nucleofectionappears to cause ˜50% non-specific knockdown of DC-Sign even in the caseof control siRNA delivery. This may indicate off-target effects due tothe electroporation treatment. i.e. exposure of cells to the electricfields resulted in damage to the cells, specifically the targetproteins, resulting in a measured reduction in expression levels in theabsence of siRNA targeting the protein. These results indicate that themembrane deformation method is more specific compared toelectroporation/nucleofection methods, which are associated withnon-specific (off-target) effects.

FIG. 19 is a series of graphs showing data pertaining to the delivery ofimpermeable alexa-488 labeled 10 kDa dextran dyes to cells in wholeblood. Fluorescently labeled dyes were mixed with whole blood, and themixture of whole blood+labeled dye ran through the device, and thenmeasured for delivery to the blood cells by FACS after RBC lysis. Theresults demonstrate that the dyes were successfully delivered into thecells and that cargo compounds that have been characterized as “cellimpermeable” are effectively delivered to immune cells using cellsqueezing. It is surprising that the delivery process works in wholeblood. Whole blood is very hard to manipulate without purification,e.g., fractionation or separation of peripheral blood mononuclear cellsfrom red blood cells yet devices disclosed herein are capable ofdelivering compounds into immune cells in whole blood well. For anon-limiting example, see FIGS. 33 and 34.

FIG. 20 is a diagram of the microfluidic membrane disruption system.

FIGS. 21A-B are dot plots showing mRNA expression one day after deliverywith a 10-4 chip at 120 psi in Optimem buffer.

FIG. 22A is a diagram and FIG. 22 B is a bar graph showing delivery of10 kDa alexa 488-labeled dextran at different pressures with differentchip angles. The number in parenthesis is the constriction angle. Chipangle range from 0-180 degrees, e.g., 11-105 degrees. The schematicindicates chip angle. Depth parameter ranges from 2 μm to 1 mm, e.g.,˜20 pm and is further described in U.S. Patent Pub. No. 20140287509(hereby incorporated by reference). Exemplary parameters include 0-30 μmlength/3-4 μm width/20 μm depth/11 degree angle fot naïve T and B cells.

FIG. 23 is a line graph showing transcription factor delivery to NKcells and pDCs. A Oct4 mRNA expression was measured 4 hours afterdelivery of Oct4 recombinant protein to splenic mouse NK cells. Thesedata indicate that the transcription factor is active and capable ofinducing expression of endogenous Oct4. The Oct4 transcription factor isone of the four factors required for iPS generation (Oct4, Klf4, cMyc,Sox2) and exercises positive feedback control on itself. Delivery ofactive Oct4 protein yields increased Oct4 mRNA expression. Delivery ofactive transcription factors is a critical step in the reprogrammingprocess for protein-based methods. These methods have many advantagesover viral and DNA based systems as they minimize the risk ofintegration.

FIGS. 24A-B are line graphs showing that no detected inhibition ofnormal DC function was observed in response to squeezing. FIG. 24A:splenic DCs cultured in LPS (1 ug/ml) after treatment by squeezing, byantigen endocytosis alone, and untreated cells showed no detectabledifference in their ability to upregulate CD80 and CD86 expression. Nonstimulated endocytosis conditions and untreated cells maintained at 4°C. were used as controls. FIG. 24B: splenic DCs from a CD45 congenicmouse were injected into the footpad of C57BL6 mice with LPS (1 ug/ml)and recovered in the draining lymph node 18 hrs post injection. Nosignificant difference in lymph node homing ability was detected.

FIGS. 25A-D are graphs showing that use of the membrane deformationdevice system and methods lead to more effective antigen presentationcompared to other antigen delivery approaches. FIG. 25A: in vivoproliferation of adoptively transferred CD8+OT-I T cells in response tosubcutaneous DC vaccination. Device treated BMDCs (0.1 mg/ml of Ova)demonstrated a significant increase (P<0.0001) in T cell proliferationrelative to those that were allowed to endocytose antigen. FIG. 25B: invitro proliferation of CD8+OT-I T cells that have been cocultured withBMDCs treated with the cell lysate of an Ova expressing melanoma cellline (B16F10). The % indicates the fraction of cell lysate material thatwas added to a BMDC cell suspension prior to treatment by a 10-6CellSqueeze device. The CD8 T cells were labeled by carboxyfluorosceinsuccinimidyl ester (CFSE) prior to contact with the APC. If theyproliferate, the CFSE dye is distributed amongst daughter cells andresults in lower fluorescence intensity per cell. Unproliferated T cellsretain high CFSE intensity. FIG. 25C: measuring the fraction ofantigen-specific IFNγ secreting CD8 T cells. In these experiments, micewere vaccinated against Ova in the absence of adoptively transferredOT-I T cells. The endogenous, antigen specific response was measured byisolating the spleens of vaccinated mice 7 days after vaccination andrestimulating them in vitro with SIINFEKL peptide (an OVA epitope).Antigen-specific CD8 T cells secrete IFNγ in response to restimulation.FIG. 25D; in vitro proliferation of CD8+OT-I T cells that have beencocultured with B cells treated with two different device designs (0.1mg/ml Ova). CpG was used as an adjuvant in these experiments.

FIG. 26 is a graph showing the results of a CFSE proliferation assay invivo: This graph is measuring the proliferation of antigen specific CD8T cells. As the CD8T cells are activated and proliferate in the mousethey dilute the CFSE dye and have a lower fluorescence intensity. Inthis case, donor T cells treated by the device or the positive controlyielded much greater activation and proliferation of CD8 T cells in therecipient mouse. Endocytosis controls by contrast show minimal effect.

FIG. 27 is a series of histograms showing proliferation of antigenspecific OT-I T cells in mice in response to vaccination with antigentreated wild type T cells. T cell proliferation responses are measuredby CFSE staining, the stain is diluted as the cells proliferate. Lowerintensities indicate greater responses, higher intensity peaks indicateno/less response. Each column represents a replicate of the experimentwith the lymph nodes and spleen derived from the same mouse. Each columnof experiments involved 3 mice (9 mice in total).

FIGS. 28A-B are histograms showing gating on both of DQ-OVA+3kDa-Dextran+ T cells.

FIG. 29 is a bar graph demonstrating the use of mouse T cells as antigenpresenting cells. The T cells, which were treated by cell squeezing toload unprocessed ovalbumin antigen, were cultured with OT-1(SIINFEKL-specific T cell line) and activation markers, CD25 and CD69evaluated.

FIG. 30 is a schematic of an exemplary procedure for making andcharacterizing squeeze-mediated production of B cells as antigenpresenting cells.

FIG. 31 is a series of histograms showing that cell-squeezed B cellsinduced potent CD8+ T cell proliferation.

FIG. 32 is a series of histograms showing that cell-squeezed dendriticcells (OVA delivered) secrete gamma interferon.

FIG. 33 shows data and a cartoon showing delivery of 10 kDa material toHuman B and T-cells directly in unmodified whole blood.

FIG. 34 is a graph showing viability and delivery efficiencies in wholeblood.

DETAILED DESCRIPTION

A vector-free microfluidic delivery platform (CellSqueeze) is used todeliver macromolecules directly into the cytosol of primary immunecells, e.g., mouse, human, with minimal cytotoxicity. The principleunderlying this approach is temporary membrane disruption by rapidmechanical deformation, or squeezing, of the target cell, which permitsthe uptake by diffusion of macromolecules in the fluid medium and isfollowed by cell membrane repair (see, e.g., U.S. Patent Publication No.20140287509, hereby incorporated by reference. Using a library ofmicrofluidic designs, uptake of test compounds such as dextran polymers,antibodies and small interfering RNAs (siRNA) were delivered intoprimary human and murine T cells, B cells, monocytes/macrophages, anddendritic cells+. The results demonstrate the utility of the platform todeliver a variety of different sizes and types of macromolecules.Efficient delivery of material to different classes of immune cells,which have a large range of cell diameter (˜8-30 μm), distinctmorphology, anisotropy and membrane flexibility, required identificationof specific conditions for different cell types. For cell squeezing,width of the constriction is a critical parameter, and other parameterssuch as geometric elements, speed, buffer, and temperature may alsoaffect delivery of cargo. Exemplary constriction widths for delivery ofcargo to naïve T or B cells are in the range of 3-4 μm; for delivery toactivated T or B cells, 4-6 μm; and for delivery to dendritic cells, 6-8μm.

Delivery of siRNAs resulted in robust gene knockdown. Moreover, deliveryof antiviral siRNAs to CD4+ T cells inhibited HIV replication,demonstrating the functional utility of microfluidic-based delivery.Similarly, delivery of antigenic proteins to dendritic cells and B cellsresulted in more effective antigen presentation and greateractivation/proliferation of antigen-specific CD8+ T cells in vitro andin vivo. By providing a platform for robust intracellular delivery withminimal loss in viability, cell squeezing represents a flexible anduseful tool to probe and control immune cell function for research andclinical applications.

The intracellular delivery of biomolecules, such as proteins and siRNAs,into primary immune cells, especially resting lymphocytes, has beendifficult. The vector-free microfluidic platform described herein causestemporary membrane disruption by rapid mechanical deformation of a cell,which leads to intracellular delivery of macromolecules to immune cellssuch as T cells, B cells, monocytes/macrophages, and dendritic cells. Alibrary of 16 microfluidic device designs was tested for the ability todeliver dextran polymers, siRNA and antibodies to human and murineimmune cells. The activity of the delivered material was verified bymeasuring siRNA-mediated knockdown of CD45, DC-SIGN, and CD4 proteins.Microfluidic delivery, which requires neither viral vectors norelectrical fields, resulted in comparable or better delivery thanelectroporation, with less cellular toxicity. The technique's utility indisease applications was shown by inhibiting HIV viral replication inprimary human CD4 T cells treated with siRNAs directed against viral vifand gag genes. Thus, vector-free microfluidic delivery provides a way toovercome the hurdle of cytosolic delivery of macromolecules to cellswhich in the past were difficult to engineer (e.g., primary immunecells). The methods and device are therefore useful for engineering ofimmune cell function.

In certain aspects, the present disclosure relates to methods forpreferentially delivering a compound to the cytosol of an immune cell,comprising passing a cell suspension comprising the immune cell througha microfluidic device and contacting the suspension with the compound,wherein the device comprises a constriction of a diameter of about 2 μm,3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm or 2 μm-10 μm andwherein the amount of compound delivered to the immune cell is at least10% greater than that delivered to a non-immune cell. In certainaspects, the present disclosure relates to method for delivering acompound to the cytosol of an immune cell, comprising passing a cellsuspension comprising the immune cell through a microfluidic device andcontacting the suspension with the compound, wherein the devicecomprises a constriction of a diameter of about 2 μm, 3 μm, 4 μm, 5 μm,6 μm, 7 μm, 8 μm, 9 μm, 10 μm or 2 μm-10 μm.

The term ‘cell-squeezed’ refers to the method comprising passing a cellsuspension through a microfluidic device comprising a constriction. Insome embodiments, the cell suspension is contacted with the compound,before, concurrently, or after passing through the microfluidic device.In some embodiments, the immune cell comprises at least 10%, 25%, 50%,2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, or more of thecompound after passing through the device as compared to an immune cellcontacted with the compound without passing through the device.

Engineering Immune Cell Function

By delivering material into the intracellular space of immune cells bytransiently disrupting or deforming membrane integrity, internalmechanisms can be interrogated and characterized, and their functionmanipulated or altered for a diversity of applications.

An effective way to engineer a cell's function and/or understand itsinner workings is introduce material (e.g. bioactive molecules) into thecell and directly manipulate intracellular processes. The methodsdescribed herein comprise advantages compared to existing or previousapproaches, which largely focus on manipulating the content of the mediathat cells are in and/or signaling through binding to surface receptors.Intracellular delivery to immune cells is a significant challenge usingexisting or previous technologies. The CellSqueeze platform describedherein delivers diverse material into immune cells and has demonstratedthe ability to influence cell function in vivo and in vitro. Thesemethods are useful to program (or re-program) immune cell function forclinical use, e.g., adoptive transfer therapies. Moreover, the methodsare useful to test and elucidate immunological mechanisms to identifydrug targets and/or diagnostics.

Aspects of the present invention relate to the surprising discovery thatcompounds can be delivered to immune cells (such as human B and T cells)while they remain within whole blood. Whole blood is difficult tomanipulate without purification, e.g., fractionation or separation ofperipheral blood mononuclear cells from red blood cells. However,devices and methods disclosed herein deliver compounds into immune cellswithin whole blood. The invention enables delivery of compounds toimmune cells without the need for separation of the immune cells fromwhole blood before passing the heterogenous mixture (immune cells,erythrocytes, plasma/serum) through a cell squeezing device. Thisremarkable result has important technical benefits, and in someinstances enables the bedside treatment of patients as well as theability to process cells in situations without access tocell-fractionation, e.g., a battlefield. For example, a subject may havewhole blood removed, processed through a device of the invention, andthen reinfused, e.g., in a continuous process. Since the isolation orenrichment of immune cells is not required, less manipulation of thecells is required, and the cells need not be processed using media suchas artificial media. Additionally, treating immune cells in whole bloodcan be performed with high efficiency while maintaining high levels ofviability. See, e.g., FIGS. 33 and 34.

In some embodiments that can be combined with the previous embodiments,the cell suspension comprises mammalian cells. In some embodiments, thecell suspension comprises a mixed cell population. In some embodiments,the cell suspension is whole blood. In some embodiments, the cellsuspension comprises buffy coat cells. In some embodiments, the cellsuspension is lymph. In some embodiments, the cell suspension comprisesperipheral blood mononuclear cells. In some embodiments, the cellsuspension comprises a purified cell population. In some embodiments,the cell is a primary cell or a cell line cell. In some embodiments, thecell is a blood cell. In some embodiments, the blood cell is an immunecell. In some embodiments, the immune cell is a lymphocyte. In someembodiments, the immune cell is a T cell, B cell, natural killer (NK)cell, dendritic cell (DC), NKT cell, mast cell, monocyte, macrophage,basophil, eosinophil, or neutrophil. In some embodiments, the immunecell is an adaptive immune cell such as a T cell and B cell. In someembodiments, the immune cell is an innate immune cell. Exemplary innateimmune cells include innate lymphoid cells (ILC1, ILC2, ILC3),basophils, eosinophils, mast cells, NK cells, neutrophils, andmonocytes. In some embodiments, the immune cell is a memory cell. Insome embodiments, the immune cell is a primary human T cell. In someembodiments, the cell is a mouse, dog, cat, horse, rat, goat, monkey, orrabbit cell. In some embodiments, the cell is a human cell. In someembodiments, the cell suspension comprises non-mammalian cell. In someembodiments, the cell is a chicken, frog, insect, or nematode cell.

In some examples, the immune cell is in a resting state compared to anactivated state, e.g., cells in an activate state generally comprise alarger diameter compared to cells of the same phenotype in a restingstate. For example, cells are characterized by expression of thefollowing markers: CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28,CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1 and CXCR5, each of which may bemodulated (increased or reduced by introducing molecules into the immunecells using the methods described). In some embodiments, the expressionof one or more markers is increased on the immune cells by delivery ofcompounds into the immune cells. In some embodiments, the expression ofone or more markers is decreased on the immune cells by delivery ofcompound into the immune cells. In some embodiments, the expression ofone re more markers is increased and the expression of one or moremarkers is decreased on the immune cells by delivery of compounds intothe immune cells. In some embodiments, the immune cell is a naïve immunecell. Naïve immune cells, e.g., T cells, are characterized bycomparatively low levels of expression of CD25, CD80, CD86, PD-1, andCTLA-4 and by comparatively high level of CCR7 (compared to activatedcells) as compared to the level of expression by activated immune cells.

Aspects of the present subject matter relate to major histocompatibilitycomplexes (MHCs). The major function of MHCs s to bind to peptidefragments derived from pathogens and display them on the cell surfacefor recognition by the appropriate T cells. In humans, MHCs are alsocalled human leukocyte antigens (HLAs). HLAs corresponding to MHC classI (HLA-A, HLA-B, and HLA-C) present peptides from inside the cell. Forexample, if the cell is infected by a virus, the HLA system bringsfragments of the virus to the surface of the cell so that the cell canbe destroyed by the immune system. These peptides are produced fromdigested proteins that are broken down in the proteasomes. In generaland with respect to MHC-1, these particular peptides are small polymers,about 8-10 amino acids in length. Foreign antigens presented by MEWclass I attract killer T-cells (also called CD8 positive- or cytotoxicT-cells) that destroy cells. HLAs corresponding to MHC class II (HLA-DP,HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR) present antigens fromoutside of the cell to T-lymphocytes. These particular antigensstimulate the multiplication of T-helper cells, which in turn stimulateantibody-producing B-cells to produce antibodies to that specificantigen. Self-antigens are suppressed by regulatory T cells.

In certain aspects, the present disclosure relates to methods fordelivering a compound or composition into a cell. In some embodiments,the compound is a single compound. In some embodiments, the compound isa mixture of compounds. In some embodiments, the compound comprises anucleic acid. In some embodiments, the compound is a nucleic acid.Exemplary nucleic acids include, without limitation, recombinant nucleicacids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA,miRNA, IncRNA, tRNA, and shRNA. In some embodiments, the nucleic acid ishomologous to a nucleic acid in the cell. In some embodiments, thenucleic acid is heterologous to a nucleic acid in the cell. In someembodiments, the compound is a plasmid. In some embodiments, the nucleicacid is a therapeutic nucleic acid. In some embodiments, the nucleicacid encodes a therapeutic polypeptide.

In some embodiments the nucleic acid encodes a reporter or a selectablemarker. Exemplary reporter markers include, without limitation, greenfluorescent protein (GFP), red fluorescent protein (RFP), auquorin,beta-galactosidase, Uroporphyrinogen (urogen) III methyltransferase(UMT), and luciferase. Exemplary selectable markers include, withoutlimitation, Blasticidin, G41 8/Geneticin, Hygromycin B, Puromycin,Zeocin, Adenine Phosphoribosyltransferase, and thymidine kinase. In someembodiments, the compound is a nucleic acid encoding for a MHC complex.In some embodiments, the compound is a nucleic acid encoding for a MHCclass I or MHC class II complex. In some embodiments, the nucleic acidencodes for a chimeric antigen receptor, such as a chimeric T cellreceptor. In some embodiments, the nucleic acid encodes for arecombinant T cell receptor. For example, nucleic acids encodingchimeric antigen receptors are introduced into a T cell in a virus-freeway, i.e., by cell squeezing, to maintain expression of CAR-T. Forexample, introduction of DNA is accomplished without the use of a viralparticle. Nucleic acid constructs may however include viral genomeelements, which may help the integration or be maintained as anextrachromosomal nucleic acid.

In some embodiments, the compound comprises a protein or polypeptide. Insome embodiments, the compound is a protein or polypeptide. In someembodiments, the protein or polypeptide is a therapeutic protein,antibody, fusion protein, antigen, synthetic protein, reporter marker,or selectable marker. In some embodiments, the protein is a gene-editingprotein or nuclease such as a zinc-finger nuclease (ZFN), transcriptionactivator-like effector nuclease (TALEN), mega nuclease, or CRErecombinase. In some embodiments, the fusion proteins can include,without limitation, chimeric protein drugs such as antibody drugconjugates or recombinant fusion proteins such as proteins tagged withGST or streptavidin. In some embodiments, the compound is atranscription factor. Exemplary transcription factors include, withoutlimitation, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORγt. Insome embodiments, the nucleic acid is a transposon. A transposon, ortransposable element, is a DNA segment that inserts itself into anotherposition within the genome.

In some embodiments, the compound comprises an antigen. In someembodiments, the compound is an antigen. An antigen is a substance thatstimulates a specific immune response, such as a cell orantibody-mediated immune response. Antigens bind to receptors expressedby immune cells, such as T cell receptors (TCRs), which are specific toa particular antigen or to antigen presentation molecules such asMHC/HLA heterodimers. Antigen-receptor binding subsequently triggersintracellular signaling pathways that lead to downstream immune effectorpathways, such as cell activation, cytokine production, cell migration,cytotoxic factor secretion, and antibody production. In someembodiments, the compound comprises a disease-associated antigen. Insome embodiments, antigens are derived from foreign sources, such asbacteria, fungi, viruses, or allergens. In some embodiments, antigensare derived from internal sources, such as tumor cells or self-proteins(i.e. self-antigens). In some embodiments, the tumor antigen is in atumor lysate. Self-antigens are antigens present on an organism's owncells. Self-antigens do not normally stimulate an immune response, butmay in the context of autoimmune diseases, such as Type I Diabetes orRheumatoid Arthritis, Multiple Sclerosis (and other demyelinatingdisorders). In some embodiments, the antigen is a neoantigen.Neoantigens are antigens that are absent from the normal human genome,but are created within oncogenic cells as a result of tumor-specific DNAmodifications that result in the formation of novel protein sequences.Exemplary viral antigens include HIV antigens, Ebola antigen, HPVantigens, and EBV antigens, which are purified or delivered as amixture, or delivered as killed or attenuated virus or virus fragments.In some embodiments, the HPV antigens are derived from the oncogenes E6and E7 of HPV16. In some embodiments, the compound comprises cell lysatefrom tissue infected with an unknown pathogen. In some embodiments, theantigen is a non-protein antigen, such as a lipid, glycolipid, orpolysaccharide.

In some embodiments the protein or polypeptide is a reporter or aselectable marker. Exemplary reporter markers include, withoutlimitation, green fluorescent protein (GFP), red fluorescent protein(RFP), auquorin, beta-galactosidase, Uroporphyrinogen (urogen) IIImethyltransferase (UMT), and luciferase. Exemplary selectable markersinclude, without limitation, Blasticidin, G418/Geneticin, Hygromycin B,Puromycin, Zeocin, Adenine Phosphoribosyltransferase, and thymidinekinase.

In some embodiments, the compound comprises a small molecule. In someembodiments, the compound is a small molecule. Exemplary small moleculesinclude, without limitation, fluorescent markers, dyes, pharmaceuticalagents, metabolities, or radionucleotides. In some embodiments, thepharmaceutical agent is a therapeutic drug and/or cytotoxic agent.

In some embodiments, the compound comprises a nanoparticle. Examples ofnanoparticles include gold nanoparticles, quantum dots, carbonnanotubes, nanoshells, dendrimers, and liposomes. In some embodiments,the nanoparticle contains or is linked (covalently or non-covalently) toa therapeutic molecule. In some embodiments, the nanoparticle contains anucleic acid, such as mRNA or cDNA. In some embodiments, thenanoparticle contains a label, such as a fluorescent or radioactivelabel.

Aspects of the present invention relate to the improved delivery ofintracellular antibodies. Non-limiting examples of intracellularantibodies are described in U.S. Pat. No. 6,004,940, issued Dec. 21,1999; U.S. Pat. No. 6,329,173, issued Dec. 11, 2001; U.S. PatentApplication Publication No. 2010/0143371, published Jun. 10, 2010; andU.S. Patent Application Publication No. 2006/0034834, published Feb. 16,2006, the contents of each of which are incorporated herein byreference. A limitation impacting the usefulness of intracellularantibodies has been expressing them in cells of interest, includingcells within whole blood. The present invention overcomes thislimitation, and enables isolated antibodies, or constructs encodingantibodies, to be delivered to the cytosol of immune cells.

The invention encompasses not only delivery of an intact monoclonalantibody, but also an immunologically-active antibody fragment, e.g., aFab or (Fab)2 fragment; an engineered single chain Fv molecule; or achimeric molecule, e.g., an antibody which contains the bindingspecificity of one antibody, e.g., of murine origin, and the remainingportions of another antibody, e.g., of human origin. In another example,the chimeric molecule is a fusion of single-chain variable fragment(scFv) derived from a monoclonal antibody fused to CD3-zetatransmembrane and endodomain. Such molecules result in the transmissionof a zeta signal in response to recognition by the scFv of its target.The variable portions of an immunoglobulin heavy and light chain arefused by a flexible linker to form a scFv. This scFv is preceded by asignal peptide to direct the nascent protein to the endoplasmicreticulum and subsequent surface expression. A flexible spacer allowsthe scFv to orient in different directions to enable antigen binding.The transmembrane domain is a typical hydrophobic alpha helix usuallyderived from the original molecule of the signaling endodomain whichprotrudes into the cell and transmits the desired signal. Such chimericantigen receptors are delivered to T cells using the microfluidicsqueeze method described herein.

In some embodiments, the compound comprises a chimeric antigen receptor(CAR). In some embodiments, the compound is a chimeric antigen receptor(CAR). In some embodiments, the CAR is a fusion of an extracellularrecognition domain (e.g., an antigen-binding domain), a transmembranedomain, and one or more intracellular signaling domains. Upon antigenengagement, the intracellular signaling portion of the CAR can initiatean activation-related response in an immune cell, such as the release ofcytokines or cytolytic molecules. In some embodiments, the CAR is achimeric T-cell antigen receptor. In some embodiments, the CAR containsan antigen-binding domain specific to a tumor antigen. In someembodiments, the CAR antigen-binding domain is a single-chain antibodyvariable fragment (scFv). In some embodiments, the compound enhances Tcell function. In some embodiments, the compound that enhances T cellfunction is an immune checkpoint pathway inhibitor. Exemplary immunecheckpoint pathway inhibitors include, without limitation, programmeddeath-1 pathway inhibitors, a programmed death ligand-1 pathwayinhibitors, and an anti-cytotoxic T-lymphocyte antigen 4 pathwayinhibitors. For example, the immune checkpoint pathway inhibitors cantarget SHP2, a tyrosine phosphatase that is involved in PD-1 and CTLA-4signaling.

In some embodiments, the compound comprises a fluorescently taggedmolecule. In some embodiments, the compound is a fluorescently taggedmolecule, such as a molecule tagged with a fluorochrome such as pacificblue, Alexa 288, Cy5, or cascade blue. In some embodiments, the compoundis a radionucleotide, dextran particle, magnetic bead, or impermeabledye. In some embodiments, the compound is a 3 kDa dextran particlelabeled with PacBlue. In some embodiments, the compound is a 10 kDadextran particles labeled with Alexa488. In some embodiments, thecompound is a small molecule fluorophore tagged protein. In someembodiments, the compound is a small molecule tagged with Alexa647. Insome embodiments, the compound comprises a virus or virus-like particle.In some embodiments, the virus is a therapeutic virus. In someembodiments, the virus is an oncolytic virus. In some embodiments, thevirus or virus-like particle contains nucleic acid encoding for atherapeutic molecule, such as a therapeutic polypeptide.

In some embodiments, the compound comprises a tolerogenic factor. Insome embodiments, the compound comprises an adjuvant. In someembodiments, the compound comprises a differentiation factor. Exemplarydifferentiation factors to be delivered to the cytosol of T cells topromote differentiation and/or activation/maturation of T cells includeT-box transcription factors T-bet and Eomesodeimin (Eomes), NFKB and/orforkhead box P3 (FOXP3).

Exemplary compounds and compositions for intracellular delivery include:

-   -   Nucleic acids, particularly: DNA (plasmid or other oligos) and        RNA (e.g. siRNA, mRNA, tRNA, saRNA, lncRNA, miRNA, guide RNA)        chemically, biologically or otherwise modified. Proteins: e.g.        antibodies, inhibitors, enzymes (e.g. kinases), transcription        factors, ribosomes, antigens, cell lysates;    -   Peptides: long (100-10,000 amino acids) and short (1-100 amino        acids) Nanomaterials: e.g. lipid-based nanoparticles, polymeric        nanoparticles, carbon nanotubes, quantum dots, metallic        nanoparticles (including gold);    -   Virus: Cytoplasmic delivery of viral (or virus-like) particles        yield successful gene delivery for cells that are otherwise        resistant to infection. Use of a replication incompetent virus        represents an additional means of manipulating cell function;    -   Other materials: polymers, dyes, TrisNTA, small molecule drugs,        adjuvants, probes;    -   Mixtures of any combination of the above.        Exemplary cell types info which compounds/compositions are        delivered (all adaptive and innate immune cells) include:    -   All mammalian species, e.g., human, mouse, dog, cat, horse,        monkey    -   B cells (e.g. naïve B cells, plasmablasts);    -   T cells (e.g. Th1, Th17, Th2, Treg, CD8, CD4, Trm, Tern, Tcm);    -   Dendritic cells (e.g. pDCs, monocyte derived DCs, cDCs, CD8⁺        DCs, CD11b⁺ DCs;    -   Monocytes, macrophage;    -   Neutrophils, NK cells, innate lymphoid cells (ILC1, ILC2, ILC3),        basophils, granulocytes and mast cells.    -   Precursor cells, (hematopeotic stem cells, CLPs, mesenchymal        stem cells)        Engineering Immune Cell Antigen Presentation

Certain aspects of the present disclosure relate to a method forengineering of immune cell function comprising intracellular delivery ofcompounds by transiently disrupting a membrane surrounding the cytoplasmof the immune cell and delivering into the cytosol an antigen. In someembodiments, the antigen comprises a length of greater than 7, 8, 9 or10 amino acids and wherein the immune cell processes the antigen anddisplays a class I histocompatibility antigen restricted processed formof the antigen on a surface of the immune cell.

Certain aspects of the present disclosure relate to a method forengineering of immune cell function comprising intracellular delivery ofa compound by passing an immune cell through a microfluidic devicecomprising a constriction and contacting the immune cell with thecompound. In some embodiments, the compound comprises an antigen and theimmune cell processes the antigen and displays the antigen on a surfaceof the immune cell. In some embodiments, the immune cell displays aclass I histocompatibility antigen restricted processed form of theantigen on a surface of the immune cell. In some embodiments, the immunecell displays a class II histocompatibility antigen restricted processedform of the antigen on a surface of the immune cell. In someembodiments, the cell membrane is disrupted by passing the immune cellthrough a constriction of a diameter of 2 μm-10 μm. In some embodiments,the antigen comprises a full-length, unprocessed protein. In someembodiments, the immune cell is contacted with an effector T cell, suchas a CD8+ T cell and activates a cytotoxic T cell immune response. Insome embodiments, the immune cell is contacted with an effector T cell,such as a CD4+ T cell, and activates a helper T cell immune response. Insome embodiments, immune cell is contacted with an effector T cell andactivates a tolerogenic T cell immune response. In some embodiments, theimmune cell comprises a B cell, dendritic cell or macrophage. In someembodiments, the immune cell comprises a T cell.

Certain aspects of the present disclosure relate to a method forconferring an antigen presenting phenotype on a T cell, comprisingdelivering a whole, unprocessed antigen to the cytosol of a T cell bypassing the T cell through a microfluidic device, wherein the devicecomprises a constriction of a diameter of 2 μm-10 μm and wherein the Tcell comprises a class I histocompatibility antigen restricted processedform of the antigen on a surface of the immune cell following passagethrough the microfluidic device. Certain aspects of the presentdisclosure relate to a method for conferring an antigen presentingphenotype on a T cell, comprising delivering a whole, unprocessedantigen to the cytosol of a T cell by passing the T cell through amicrofluidic device, wherein the device comprises a constriction of adiameter of 2 μm-10 μm and wherein the T cell comprises a class IIhistocompatibility antigen restricted processed form of the antigen on asurface of the immune cell following passage through the microfluidicdevice. In some embodiments, the antigen comprises a tumor antigen or aviral antigen. In some embodiments, the T cell is further contacted witha second T cell, the second T cell comprising a class Ihistocompatibility antigen restricted cytotoxic T cell phenotype. Insome embodiments, the T cell is further contacted with a second T cell,the second T cell comprising a class II histocompatibility antigenrestricted helper T cell phenotype.

Certain aspects of the present disclosure relate to the use of acell-squeezed, antigen loaded T cell to activate a cytotoxic T cellresponse specific for an antigen. Certain aspects of the presentdisclosure relate to the use of a cell-squeezed, antigen loaded T cellto activate a helper T cell response specific for an antigen. Certainaspects of the present disclosure relate to the use of a cell-squeezed,antigen loaded T cells to induce a tolerogenic T cell response specificfor an antigen.

Engineering Immune Cell Homing

Certain aspects of the present disclosure relates to a method forconferring a horning phenotype to an immune cell, comprising deliveringa compound to the cytosol of a T cell by passing the immune cell througha microfluidic device, wherein the device comprises a constriction of adiameter of 2 μm-10 μm and wherein the compound confers the expressionof a horning phenotype to the immune cell. For example, the deliveredcompounds can increase the expression of chemokine receptors that directhorning to a particular site and downregulate the expression ofconflicting chemokine receptors.

In some embodiments, the compound comprises a nucleic acid. In someembodiments, the compound is a nucleic acid. Exemplary nucleic acidsinclude, without limitation, recombinant nucleic acids, DNA, recombinantDNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, IncRNA, tRNA,and shRNA.

In some embodiments, the compound comprises a protein or polypeptide. Insome embodiments, the compound is a protein or polypeptide. In someembodiments, the protein is a gene-editing protein or nuclease such as azinc-finger nuclease (ZFN), transcription activator-like effectornuclease (TALEN), mega nuclease, or CRE recombinase. In someembodiments, the compound is a transcription factor. Exemplarytranscription factors include, without limitation, OctS, Sox2, c-Myc,Klf-4, T-bet, GATA3, FoxP3, and RORγt. In some embodiments, thetranscription factor induces the cellular expression of MHC complexes.

In some embodiments, the compound comprises a chimeric antigen receptor(CAR). In some embodiments, the compound is a chimeric antigen receptor(CAR). In some embodiments, the CAR is a fusion of an extracellularrecognition domain (e.g., an antigen-binding domain), a transmembranedomain, and one or more intracellular signaling domains. Upon antigenengagement, the intracellular signaling portion of the CAR can initiatean activation-related response in an immune cell, such as homing to aparticular tissue or physiological location. In some embodiments, theCAR is a chimeric T-cell antigen receptor. Engineering Immune Cells forTolerance

Certain aspects of the present disclosure relates to a method forconferring a tolerogenic phenotype to an immune cell, comprisingdelivering a compound to the cytosol of a T cell by passing the immunecell through a microfluidic device, wherein the device comprises aconstriction of a diameter of 2 μm-10 μm and wherein the compoundinduces the differentiation of the immune cell into a cell with atolerogenic phenotype. In some embodiments, the compound comprises anucleic acid. In some embodiments, the compound is a nucleic acid.Exemplary nucleic acids include, without limitation, recombinant nucleicacids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA,miRNA, lncRNA, tRNA, and shRNA.

Certain aspects of the present disclosure relate to a method of treatinga patient by introducing the immune cells modified according to themethods described to a patient. In some embodiments, the immune cellsare for use in immunosuppressive therapy. In some embodiments, the cellsare isolated from a patient, modified according to the methods describedherein, and introduced back into the patient. In some embodiments, animmune checkpoint inhibitor is further administered to the patient.

Engineering Kamikaze Immune Cells

Certain aspects of the present disclosure relates to a method forgenerating a Kamikaze immune cell, comprising delivering self-amplifyingRNA to the cytosol of a T cell by passing the immune cell through amicrofluidic device, wherein the device comprises a constriction of adiameter of 2 μm-10 μm and wherein the self-amplifying RNA encodes forcontinual production of an encoded protein. In some embodiments, thecompound comprises a nucleic acid. In some embodiments, the compound isa nucleic acid. In some embodiments, the nucleic acid is self-amplifyingRNA (saRNA).

Screening Antigens for Vaccine Development

In some embodiments, immune cells modified according to the methodsdescribed herein are used to screen antigens for vaccine development.For example, a tumor cell lysate is delivered to antigen presentingcells, T cells, or B cells using the squeeze method described herein.The APC are incubated with patient-derived T cells or T cellclones/lines to determine the identity of vaccine candidate antigens. Inanother approach, the antigen processed and presented by thetumor-lysate loaded APCs is identified by Mass Spectroscopy. Antigensidentified in this manner are then subsequently used for vaccination.

Immune Cell Trafficking

Certain aspects of the present disclosure relate to a method ofdetermining T cell trafficking within a patient, comprising delivering alabel into a T cell according to the methods described herein andadministering the labeled T cell into a patient, wherein T celltrafficking within the patient can be determined by detecting thelabeled T cell. In some embodiments, The label is a fluorescent orradioactive label. In some embodiments, the T cell trafficking istrafficking to a tumor. For example, T cells can be isolated from apatient, passed through the microfluidic device in order to deliver anisotope into the T cells, and injected back into the patient. Imagingmethods, such as a PET scan, can then be used to detect the label andtrack the trafficking of the T cells through the body.

Antigen Presentation for Vaccines

Because the system enables delivery of proteins preferentially to immunecells, it is useful to engineer such cells for use in vaccination of apatient (human, mouse, non-human primate, etc.) against a target ofinterest. By delivering specific antigenic proteins (or mixtures ofantigenic proteins, mixtures of proteins+adjuvant, or peptidescorresponding to fragments of the proteins) directly to the cytosol of atarget cell (e.g., a DC, T cell or B cell), MHC class I presentation ofan antigen is induced which subsequently drives CD8 T cell mediatedimmunity against a target disease, e.g., cancer or a pathogenicmicrobial infection such as a viral infection. The optional use ofadjuvants in this process enhances the response (e.g. co-delivery of amaterial that enhances efficacy or incubation of cells in presence of anadjuvant factor). The ability to manipulate immune cells, which prior tothe invention, have been difficult or impossible to engineer, permitstherapeutic and prophylactic vaccine applications which were notpossible prior to the invention, especially for currently challengingdiseases such as cancer and HIV. Other manifestations includeco-delivering materials to enhance cell survival, so that the cells canpresent antigen for longer; vaccination against multiple antigenssimultaneously; vaccination in conjunction with the delivery of (orexposure to) activating factors to provide adjuvant effects and enhancethe immune response; and/or rapid-response vaccines using cell lysate asthe antigen source. In the latter example (vaccination against a newunknown pathogen/disease), infected or cancerous cells are taken from apatient, e.g. by tissue sampling or biopsy, and the lysate of thesecells is delivered to immune cells using the strategy described above.This approach raises an immune response against antigens associated withthat unknown disease without one knowing a priori the identity of theantigens.

Vaccine Adjuvants

Adjuvants or immune response activators/potentiators are used to boostelicitation of an immune cell, e.g., T cell, response to a vaccineantigen. In some embodiments, an immune cell is contacted with anadjuvant after the immune cell passes through the microfluidic device.For example the cell is contacted with the adjuvant about 5 minutes toabout 2 hours after passing through the microfluidic device or any timeor range of times therebetween. For example, the cell is contacted withthe adjuvant about 5 minutes to about 1.5 hours, about 5 minutes toabout 1 hour, about 5 minutes to about 45 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 15 minutes, or about 5minutes to about 10 minutes after passing through the microfluidicdevice. In some embodiments, the cell is contacted with the adjuvantabout 10 minutes to about 2 hours, about 15 minutes to about 2 hours,about 30 minutes to about 2 hours, about 45 minutes to about 2 hours,about 1 hour to about 2 hours, or about 1.5 hours to about 2 hours afterpassing through the microfluidic device. In addition to classicadjuvants such as alum or water-in-oil emulsions (e.g., Freund'sIncomplete Adjuvant and MF59®), other adjuvants such as ligands forpattern recognition receptors (PRR), act by inducing the innateimmunity, targeting the APCs and consequently influencing the adaptiveimmune response. Members of nearly all of the PRR families are targetsfor adjuvants. These include Toll-like receptors (TLRs), NOD-likereceptors (NLRB), RIG-I-like receptors (RLRs) and C-type lectinreceptors (CLRs). They signal through pathways that involve distinctadaptor molecules leading to the activation of different transcriptionfactors. Transcription factors (NF-κB, IRF3) induce the production ofcytokines and chemokines that play a key role in the priming, expansionand polarization of the immune responses. Activation of some members ofthe NLR family, such as NLRP3 and NLRC4, triggers the formation of aprotein complex, called inflammasome, implicated in the induction of thepro-inflammatory cytokines IL-1β and IL-18. The NLRP3 and NLRC4inflammasomes have been involved in the innate immunity induced bycertain adjuvants but their mechanism of action remains unclear.

Natural ligands or synthetic agonists for PRRs, either alone or withvarious formulations. PRR activation stimulate the production ofpro-inflammatory cytokines/chemokines and type I IFNs that increase thehost's ability to eliminate pathogens. The incorporation of pathogensassociated molecular patterns (PAMPs) in vaccine formulations improvesand accelerates the induction of vaccine-specific responses. Used incombination with alum or classical emulsion adjuvants, PAMPS are usefulto drive an immune response towards a Th1 response.

TLR3 and RLR Ligands. Double-stranded RNA (dsRNA), which is producedduring the replication of most viruses, is a potent inducer of innateimmunity. Synthetic analogs of dsRNA, such as poly(I:C) are useful asadjuvants. They act through TLR3 and RIG-I/MDA-5, inducing IL-12 andtype I IFNs production, facilitating antigen cross-presentation to MHCclass II molecules, and improving generation of cytotoxic T cells.

TLR4 Ligands. Bacterial lipopolysaccharides (LPS), which are ligands forTLR4, have long been recognized as potent adjuvants, but their pyrogenicactivity prevented their clinical use. The development of less toxicderivative includes monophosphoryl lipid A (MPLA). MPLA is useful as anadjuvant and to drive the immune response to a Th1 response.

TLR5 Ligands. The TLR5 ligand, bacterial flagellin, is a potent T-cellantigen and has potential as a vaccine adjuvant. Unlike other TLRagonists, flagellin tends to produce mixed Th1 and Th2 responses ratherthan strongly Th1 responses. Flagellin can be used as an adjuvant mixedwith the antigen or fused to a recombinant vaccine antigen.

TLR7/8 Ligands. These ligands, specialized in the recognition of singlestranded viral RNA, are also useful vaccine adjuvants. For example,Imidazoquinolines (i.e. imiquimod, gardiquimod and R848) are syntheticcompounds that activate TLR7/8 in multiple subsets of dendritic cellsleading to the production of IFN-α, and IL-12 thus promoting a Th1response.

TLR9 Ligands. Oligodeoxynucleotides containing specific CpG motifs (CpGODNs such as ODN 1826 and ODN 2006) are recognized by TLR9. They enhanceantibody production as well as drive/promote Th cell responses to Th1and away from Th2 responses.

NOD2 Ligands. Fragments of bacterial cell walls, such as muramyldipeptide (MDP), are well known adjuvants. MDP triggers the activationof NOD2 and the NLRP3 inflammasome.

These classes of adjuvants, e.g., modulators of PRR pathways, are usefulin vaccines due to their ability to induce strong cell-mediatedimmunity. Preferred adjuvants include CpG oligodeoxynucleotide, R848,lipopolysaccharide (LPS), rhIL-2, anti-CD40 or CD4O L, IL-12, and/ordicyclic nucleotides.

Engineering T Cells for Immunotherapy

Certain aspects of the present disclosure relate to a method of treatinga patient by introducing the immune cells modified according to themethods described to a patient. In some embodiments, the immune cellsare for use in immunotherapy. For example, by enabling delivery of adiversity of material to T cells, T cell function is engineered totarget a disease of interest. For example, by delivering chimericantigen receptors, or DNA/mRNA for a TCR that targets the antigen ofinterest, T cells that are specific against a disease antigen and prompta killer (and or helper) T cell response are generated. Other materials,such as NF-kB, Bcl-2, Bcl-3, Bcl-xl, upregulators of CD3/CD28, CpG,R848, suppressors of PD-1, suppressors of PDL-1, suppressors of CTLA-4are delivered to enhance cell activity and survival.

For example, one common challenge in current adoptive T cell transfertherapies is that the activated T cells are exposed to theimmunosuppressive microenvironment of the tumor and becomeexhausted/anergic thus minimizing their efficacy. Intracellular deliveryusing the methods described are used to disrupt immunosuppressionpathways [e.g. by deletion of immunosuppressive genes CTLA-4, PD-1,PD-2, PD1-1, PD1-2, or siRNA mediated knockdown, or small moleculeinhibitor/antibody based suppression using known methods such asTranscription activator-like effector nucleases (TALENS), or zinc fingernuclease (ZFN) based approaches)] and thus allow these T cells to retaintheir highly activated, killer state in the tumor environment. Moreover,this approach is used to induce T cells to become memory cells byco-delivery of appropriate factors to drive differentiation to thatphenotype (thus providing better long-term protection). In conventionaladoptive transfer, the T cells being introduced into the patient arealready in an activated, semi-exhausted state due to proliferation. Themethods described herein are used to reset their phenotype to a naïvestate. Thus, the cells become capable of much more in vivo proliferationpost-transfer and no longer have an exhausted phenotype.

In some embodiments, the methods of the present disclosure are used togenerate antigen specific T cells ex vivo. For example, antigen isdelivered to an immune cell, such as a DC, and the antigen loaded immunecell is then cultured with patient-derived T cells to activate them invitro. These T cells can then be expanded through further stimulationbefore being re-injected into the patient.

Antigen Presentation for Tolerance:

To induce tolerance to the cell-presented antigen, the cell is furthercontacted with a tolerogen such as thymic stromal lymphopoietin,dexamethasone, vitamin D, retinoic acid, rapamycin, aspirin,transforming growth factor beta, interleukin-10, or vasoactiveintestinal peptide together with antigen(s) one could induce toleranceinstead.

Tumors and T Cell Tolerance

In the tumor microenvironment, tumor reactive T cells can becometolerized. This is due to multiple suppressive mechanisms, including thetolerogenic activity of other cells associated with tumor development(Anderson et al., J Immunol 2007, 178:1268-1276; Probst et al., NatImmunol 2005, 6:280-286). Antibody-based drugs that block signalingthrough checkpoint receptors, such as CTLA-4 and PD-1, have yieldedanti-tumor responses in both primary and metastatic disease. Malignanttumors that responded to checkpoint blockade have high mutationfrequencies and are infiltrated by T-cells reactive to cancer antigens(Taneja, J Urol 2012, 188:2148-2149; Brahmer et al., N Engl J Med 2012,366:2455-2465; Wolchok et al., N Engl Med 2013, 369:122-133). While thisapproach has been successful for certain indications, the existence ofmultiple inhibitory checkpoint surface receptors can undermine theapplication of the currently limited panel of function blockingantibodies available for immunotherapy. Furthermore, the requirement forsystemic treatment with multiple blocking antibodies can have increasedtoxicity (Ribas et al., N Engl J Med 2013, 368:1365-1366; Weber et al.,Cancer 2013, 119:1675-1682), especially when used in combination(reviewed in Postow et al., J Clin Oncol 2015, 33:1974-1982 and Gao etal., Oncogene 2015). In some aspects of the invention, dramaticimprovements in patient outcomes are achieved by suppressing inhibitionpathways in tumor reactive T cells only. In non-limiting examples, thismay be achieved by inhibiting or enabling genetic knockdown or knockoutof inhibitory pathways within T-cells used in adoptive transferapproaches, such as tumor infiltrating lymphocytes (TIL), recombinantTCRs, and chimeric antigen receptor (CAR) T cells.

SHP2 is a ubiquitous tyrosine phosphatase that, upon activation inT-cells, dampens TCR signaling, and in turn the T-cell response againstcancer cells. Signaling through SHP2 by several immune checkpointreceptors diminishes T-cell activity. Inhibitory receptors that activateSHP2 include, but are not limited to, PD-1 (Yokosuka et al., J Exp Med2012, 209:1201-1217), CTLA-4 (Marengere et al., Science 1996,272:1170-1173), BTLA (Watanabe et al., Nat Immunol 2003, 4:670-679) andLAIR-1 (Lebbink et al., J Immunol 2004, 172:5535-5543) (also reviewed inNirschl et al., Clin Cancer Res 2013, 19:4917-4924). In someembodiments, the genetic inactivation or down-regulation (for example,using RNA interference) of SHP2 in tumor-reactive T cells provides anantitumor response analogous to blocking signaling of several inhibitorycheckpoint receptors. Such embodiments may have advantages (such asreduced side effects) over the therapeutic use of sodium stibogluconate(SSG), a pharmacological inhibitor of tyrosine phosphatases, includingSHP-1 and -2, in combination with other anti-tumor immunotherapies (Yiet al., Oncotarget 2011, 2:1155-1164; Naing et al., J Cancer 2011,2:81-89; Pathak et al., J Immunol 2001, 167:3391-3397). SHP2 has beenshown to play T cell extrinsic roles, and the inhibition of thismolecule on T-cells specifically could eliminate any potential sideeffects associated with a systemic inhibition of its function. Moreover,by targeting multiple inhibitory signaling pathways through geneticdisruption or down-regulation of a single intracellular signalingmolecule in T cells, greater efficacy than combinations of T celladoptive transfer and system checkpoint blockade are achieved. In someembodiments, proteins or nucleic acids, e.g., siRNA, a small moleculeinhibitor, an antibody, a Transcription activator-like effectornuclease, or a zinc finger nuclease, are delivered to immune cells,e.g., T cells, to modulate expression of a gene or activity of a geneproduct such as SHP2 to modify the behavior or function of the T cell.For example, Shpt impaired CD8 T-cells have higher potency ofcontrolling tumor progression than non-impaired cells.

The challenges of modulating gene expression in T cells associated withearlier approaches have been overcome using CellSqueeze devices andmethods described herein. Non-limiting aspects of CellSqueeze devicesare discussed in the Proceedings of the National Academy of Sciences(Sharei et al., Proc Nail Acad Sci USA 2013, 110:2082-2087) and NanoLetters (Lee et al., Nano Lett 2012, 12:6322-6327), the entire contentsof each of which are hereby incorporated herein by reference. Forexample, CellSqueeze technology may include a microfluidic chip capableof rapidly deforming cells as they pass through a constriction totemporarily disrupt their membrane and enable transport of the targetmaterial to the cell cytoplasm. Moreover, by eliminating the need forelectrical fields (e.g., in some embodiments, the device and method donot include exposure or application of the cells to an electric field)or potentially toxic exogenous materials, CellSqueeze technologyminimizes the potential for cell toxicity and off-target effects. Themicrofluidic designs and treatment processes described herein arecapable of generating more effective engineered T cell therapies for avariety of cancer indications.

Engineering T Cells for Immunosuppression

Auto-immune diseases often involve self-reactive immune cells that aredamaging healthy tissue. Intracellular delivery of FoxP3 (and/or otherfactors) to T cells is used to generate regulatory T cells to counterauto-immunity. These Tregs are generated for broad systemicimmunosuppression or to reprogram a self-antigen specific T cell to aTreg phenotype. The latter results in the Treg homing to the same targetsite as the cells that are perpetuating the auto-immunity and inducelocalized suppression of their activity.

Self-Amplifying RNA (sa RNA)

Self-amplifying RNAs provide unique capabilities, because not only dothey express the protein of interest, but they also replicate theirsequence cytoplasmically with no risk of integration into the hostgenome. Thus introduction of self-amplifying RNA into immune cells isuseful to engineer immune cell function. Some specific manifestationsinclude Kamikaze immune cells, alternatives to protein delivery, orcontinual modulation of cell function, each of which is described below.

Kamikaze Immune Cells

For vaccination or immunosuppression strategies similar to thosedescribed above, saRNAs that encode antigen(s) of interest are deliveredto immune cells that home to desired locations. For example, delivery ofa cancer antigen encoding saRNA to T cells (or B cells, monocytes, DCs,or macrophages) and injection of those cells into the patient, resultsin rapid production of the antigen in the T cells and eventual death ofthese T cells due to rapid saRNA replication. These bursts of dying Tcells loaded with target antigen simulate an infection and result inuptake of material by innate cells in the target tissues and prime avaccine response against the target antigen. Depending on the targettissue, presence of adjuvants, and overall inflammatory state of thepatient, this strategy is used to induce tolerance as well. To inducetolerance, the aforementioned kamikaze cells are engineered to releasetolerogenic factors and antigen or are contacted with tolerogenicfactors such as TGF-beta and IL-10. For example, a kamikaze T or B cellmay be loaded with an mRNA, DNA or saRNA that overexpresses a desiredantigen while also expressing factors that are tolerogenic (e.g.,secretion of TGF-beta and IL-10). These cells will then migrate to thelymphoid organs before undergoing death, which will release the antigensand the tolerogenic factors to the environment and help induce toleranceagainst the target antigen. This would help eliminate auto-reactiveeffector T cells and spur production of T regs capable of protectingagainst autoreactivity. In one non-limiting example, this approach isemployed using a rheumatoid arthritis antigen.

Alternative to Protein Delivery

saRNAs, mRNAs or expression vectors, e.g., plasmids, provide continualprotein production for the duration of vaccine treatment vs. a pulse ofprotein delivery

Continual Modulation of Cell Function

Using T cell adoptive transfer therapies as an example, development ofbetter T cell therapies is accomplished by using saRNA that encodeinhibitors of immunosuppression pathways. saRNA is also used to expressstimulatory proteins to maintain high states of T cell activation and/oranti-apoptotic proteins to prolong survival. Non-limiting examples ofsuppression inhibitors include any materials that block PD-1, PD-L1,CTLA-4 or other checkpoint inhibitors. Be expressing IL-2, NF-Kb, IL-7,IL-15, IL-12, etc. high T cell activity can be maintained and proteinslike Bcl2 and Bclx1 can help prolong survival.

Reprogramming of Immune Cell Function

Immune cells are used as a source of autologous cells. The cells arereprogrammed to perform numerous disease treating functions. Forexample, in the case of arthritis, materials are delivered to a T cellto induce its expression of a joint homing pheonotype and program therelease of factors (such as IL-4, IL-6, IL-10, IL-13, IL-11, TGFb,retinoic acid, and checkpoint stimulants) that can alleviate symptoms orin the case of Parkinson's disease, materials to a T cell or B cell toinduce its expression of brain-horning receptors and secretion offactors to improve patient outcomes. With respect to arthritis, removingpro-inflammatory cytokines is also useful, e.g., a T cell with highaffinity for IL-2 would soak up cytokine that the auto-reactive effectorcells require.

The following materials and methods were used to generate the datadescribed herein.

CellSqueeze Microfluidic Devices

The CellSqueeze platform consists of three major components: a) asilicon and glass microfluidic chip that contains multiple channels inparallel, each containing at least one constriction point b) a reservoirsystem that interfaces with the chip and allows one to load/collect thecell suspension c) a pressure regulation system to pressurize thereservoirs and facilitate fluid flow through the chip. In a typicalworkflow (FIG. 1A), the target delivery material is mixed with thedesired cells (in suspension) and load them into the reservoir. Thenpressure tubing is connected to the reservoir and the chamber ispressurized at the desired level to initiate fluid flow. Aftertreatment, the cells may be collected from the output reservoir andincubated at the desired temperature for a period of time, e.g., atleast 1, 2, 3, 4, 5 min. or more, to ensure proper membrane recoverybefore further processing.

CellSqueeze devices and the associated operating equipment were obtainedfrom SQZ Biotechnologies, USA. Devices were assembled and used inaccordance with manufacturer protocols. Sharei, A., N. Cho, S. Mao, E.Jackson, R. Poceviciute, A. Adamo, J. Zoldan, R. Langer, and K. F.Jensen. 2013. Cell squeezing as a robust, microfluidic intracellulardelivery platform. Journal of visualized experiments: JoVE: e50980.

For example, individual CellSqueeze devices and the associated reservoirsystems were kept in 70% ethanol to maintain sterility. For eachexperiment, the desired CellSqueeze device was connected to thereservoirs and 70 ul of PBS was used to flush the system prior to usewith cell samples.

During a delivery experiment, the target cells, device+reservoir, andcollection plate are kept on ice (T cells and B cells) or at roomtemperature (dendritic cells). Cells (at a concentration of 2×10⁶-1×10⁷cells/ml in PBS or culture media) are mixed with the target deliverymaterial at the desired concentration prior to being added to the fluidreservoir. The pressure tubing is connected, system is set at thedesired operating pressure, and the flow is initiated by pressurizingthe reservoir containing the sample. After passing through the chip,cells are collected from the collection reservoir and transferred to a96-well plate. This process is optionally repeated. To minimizeclogging, the direction of flow in the chip is alternated betweensamples. Samples are allowed to incubate on ice for 5 min post-treatmentbefore media is added and they are transferred for further processing.

CAR T Cells

By modifying T cells to express a chimeric antigen receptor (CAR) thatrecognizes cancer-specific antigens, one can prime the cells torecognize and kill tumor cells that would otherwise escape immunedetection. The process involves extracting a patient's T cells,transfecting them with a gene for a CAR, then reinfusing the transfectedcells into the patient.

These artificial T cell receptors (also known as chimeric T cellreceptors, chimeric immunoreceptors, or CARs) are engineered receptors,which graft an arbitrary specificity onto an immune effector cell.Typically, these receptors are used to graft the specificity of amonoclonal antibody onto a T cell. Prior to the invention, transfer ofnucleic acid coding sequence was typically facilitated by retroviralvectors. The methods described herein do not utilize or encompass viralvectors. The coding sequence or protein CAR is delivered to the cytosolof an immune cell such as a T cell using cell squeezing with thedescribed device without the need for a viral vector.

For therapeutic applications, a patient's T cells are obtained (andoptionally enriched or purified) from peripheral blood and modified toexpress an artificial (chimeric) receptor specific for a particularcancer-associated antigen. After the modification, the T cells recognizeand kill cancer. For example, an exemplary CAR recognizes CD19, anantigen expressed in B-cell¬blood malignancies. After the T cells havebeen modified to express the CAR, the modified T cells are reinfusedinto the patient. The engineered cells recognize and kill cancerouscells. Such therapy has been used for ALL, non-Hodgkin's lymphoma, andchronic lymphocytic leukemia (CLL), and is appropriate for therapy forany type of cancer, including blood-born cancers such as leukemias,B-cell malignancies (e.g., acute lymphoblastic leukemia (ALL) andchronic lymphocytic leukemia), as well as solid cancers. The cellprocessing methods described herein represent a superior process forgenerating CAR T cells.

The autologous T cells express CAR proteins that confer upon theengineered T cells the ability to recognize a specific antigen on tumorcells. Such tumor-associated antigens have been identified and are knownin the art (see tables below). The engineered CAR T cells are thenexpanded in the laboratory, and the expanded population of CAR T cellsis then infused into the patient. The T cells multiply in the patient'sbody and, recognize, bind to, and kill cancer cells that bear thetumor-associated antigen on their surfaces. Optionally, immunecheckpoint inhibitors such as programmed death-1 (PD-1) inhibitors orinhibitors of the ligand (PD-L1) and/or anti-cytotoxic T-lymphocyteantigen 4 anti-CTLA4 drugs may be combined with CAR T cells.

Treatment of Tumors

Immune cells treated as described above to introduce compounds orcompositions into the cytosol are used to treat tumors, e.g., byeliciting a tumor-specific T-cell mediated immune response to kill orinhibit the proliferation of a tumor. The method is applicable to anytumor type, because the devices and methods introduce into the immunecells tumor-specific/tumor associated antigens or mixtures thereof,e.g., tumor biopsy cell lysate preparations. For example, tumor typesinclude bladder cancer, breast cancer, colon and rectal cancer,endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma,non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroidcancer, which are prevalent in the U.S. population. (American CancerSociety: Cancer Facts and Figures, 2015, Atlanta, Ga.: American CancerSociety, 2015. Available online.) Other tumor types to be treated usingthe processed immune cells include brain (glioblastoma), liver(hepatocellular carcinoma) as well as metastatic cancers that occur inanatomic sites or tissues in the body distinct from the site or tissueof a primary tumor.

In preferred embodiments, the tumor is a pancreatic cancer, ovariancancer, melanoma, lung cancer, glioma or glioblastoma tumor. In someembodiments, the tumor of a specific patient is targeted. For example,tumor lysate from a patient may be used as antigens to be delivered toimmune cells using cell squeezing.

Tumor Cell Antigens

Purified tumor-associated antigens or tumor cell lysates (heterogenousmixture of antigens) are used as antigens to be delivered to immunecells using cell squeezing. Tumor cell lysates are produced from tumorcell lines or tumor biopsy tissue obtained from the subject, who hasbeen diagnosed with a tumor and/or is slated for treatment for apathological malignancy. Production of tumor cell lysates is known inthe art. Such tumor lysate preparations are suitable for use in cellsqueeze-based delivery to the cytosol of immune cells

For example, tumor tissue is resected from a subject, and the tissueminced. Tumor cells are processed, e.g., fractionated, enriched orpurified/isolated. In the case of non-solid tumor, blood-borne tumor(primary or metastatic), or cell line, cells enriched), cells areobtained from a bodily fluid, e.g., peripheral blood, and optionallyenriched or purified to concentrate tumor cells from non-tumor cells.Tumor cells (or populations of cells enriched for tumor cells) areprocessed to obtain a tumor cell lysate (e.g., as described by Hatfieldet al., J Immunother. 2008 September; 31(7): 620-632.) For example, thecells are subjected to a freeze-thaw cycle (e.g., 1, 2, 3, 4, 5, 10 ormore cycles of freeze/thaw). Optionally, steps include removal of soliddebris and filtering, e.g., 0.2 micron filter, to obtain a mixture ofpatient-specific tumor cell antigens.

In a non-limiting example, the cells are lysed through multiple (forexample 4) consecutive freeze/thaw cycles using liquid nitrogen. Thecells are then sonicated for about 10, 15, or 20 seconds beforecentrifuging at 1000 g to remove insoluble debris. The supernatant isthen used for lysate delivery. Optionally, lipids and/or nucleic acidsare removed from the lysate prior to delivery to immune cells. Adjuvantsare optionally added prior to delivery to augment APC function.

In some embodiments, common endogenous proteins such as actin areremoved so as to selectively increase the proportion of cancer antigensin the lysate. In some implementations, a mild surfactant is added to alysed cell composition to help prevent proteins from forming aggregatesthat may be difficult to deliver or process for presentation by a cell.

Autologous tumor cell lysate may also be generated using commerciallyavailable devices/methods, e.g., gentleMACS™ Dissociator (MiltenyiBiotec GmbH).

Tumor-associated antigens are known in the art and such antigens can bebiochemically purified, recombinantly expressed, and/or otherwisepurified/isolated. Examples of such antigens are shown in the tablesbelow.

Normal tissue Type of tumor distribution Shared Antigens Cancer-testisBAGE melanoma, spermatocytes/ (CT) Ags GAGE lymphoma, spermatogonia MAGElung, of testis, NY-ESO- bladder, colon placenta, 1 and breast ovarycells SSX carcinomas Differentiation Gp100 melanoma, prostatemelanocytes, Ags Melan-A/ cancer, colon and epithelial Mart-1 breastcarcinomas tissues, Tyrosinase prostate, PSA colon CEA Mammaglo- bin-AOverexpressed p53 esophagus, liver, ubiquitous Ags HER-2/neu pancreas,colon, (low level) livin breast, ovary, survivin bladder and prostatecarcinomas Unique Antigens Unique Ags β-catenin-m melanoma, non- N/Aβ-Actin/4/m small cell lung Myosin/m cancer, renal HSP70-2/m cancerHLA-A2- R170J Unique/Shared Antigens Tumor-associated GM2 melanoma,epithelial Carbohydrate Ags GD3 neuroblastoma, tissues MUC-1 colorectal,(e.g., renal, sTn lung, breast, intestinal, globo-H ovarian andcolorectal) prostate cancer Tumor Antigen Acute Myelocytic WT1, PR1Leukemia Breast E75, p53; HER-2/neu Colorectal ras; CEA Liver AFP; CEALung URLC10; ras; HER-2; VEGFR1 and 2; mutant p53 Melanoma MAGE; gp100;MART-1; Tyrosinase; NY-ESO-1 Ovarian P53; NY-ESO-1; HER-2 Uterine HPV16E7; Survivin; mutant p53 Pancreas ras; VEGFR1 and 2; MUC-1; Survivin(Buonaguro et al., Clin Vaccine Immunol. 2011 January; 18(1): 23-34.)

Other purified tumor antigens are known in the art, e.g., described inTumor-Associated Antigens. Edited by Olivier Gires and Barbara Seliger,2009, WILEY-VCH Verlag GmbH & Co., KGaA, Weinheim.

Viral Antigens

Virus-associated antigens may be delivered to immune cells using cellsqueezing. Virus-associated antigens are known in the art and suchantigens can be biochemically purified, recombinantly expressed, and/orotherwise purified/isolated. Examples of viral antigens are shown in thetable below.

Virus Antigen Human Viral capsid p24 protein; Group- ImmunodeficiencySpecific Antigens Virus (HIV) Human Papillomavirus Ll capsid protein;E6; E7 (HPV) Epstein-Barr Virus Viral Capsid Antigen (VGA); Early (EBV)Antigen (EA); Nuclear Antigen (EBNA) Ebola Virus Nucleoprotein (NP);Viral Proteins VP40; Viral Protein VP24; Protein VP30; Protein VP35;glycoprotein (GP) Influenza Viruses Hemagglutinin (HA) proteins, such asany of H1 to H18; neuraminidase (NA) proteins Measles Virus Haemolysin;Haemagglutinin Hepatitis C (HCV) Core Antigen Smallpox VirusIntracellular mature virion (IMV) antigen A27L; Intracellular maturevirion (IMV) antigen LIR; Extracellular enveloped virion (EEV) antigenA33R; Extracellular enveloped virion (EEV) antigen B5R Herpes SimplexVirus Glycoprotein G-1; Glycoprotein G-2; (HSV) Glycoprotein E;Glycoprotein D; Glycoprotein I; Glycoprotein B; VHS; VP7; VP16; VP21;VP23; VP24; VPAP; DNB; VP11/12; VP22a; RIR1 Severe Acute Respiratory Mprotein; E Protein; S Protein; Syndrome (SARS) Nucleocapsid ProteinAssociated Coronavirus Poliovirus VP1; VP2; and VP3Bacterial Antigens

Bacterial antigens may also be delivered to immune cells using cellsqueezing. In preferred embodiments, the bacterial antigens areassociated with intracellular bacteria such as a Mycoplasma sp.,Mycobacterium sp. (e.g., M. tuberculosis) or Listeria monocytogenes.

The following materials and methods were used to generate the datadescribed herein.

Mouse Immune Cell Isolations

T and B cells were isolated from the spleens of wild-type C57BL6/J miceusing known methods, e.g., cell-specific isolation kits from StemcellTechnologies (Vancouver, Canada) based on manufacturer's instructions(negative selection technique). Monocytes/macrophages were isolated fromthe peritoneal cavity of wild-type C57BL6/J mice 3 days followingintraperitoneal injection of 1 ml of thioglycollate solution. Cells werepurified using CD11 b positive selection kit from Stemcell Technologies(Vancouver, Canada) based on manufacturer's instructions. Cells werecultured in glutamine containing RPMI 1640 media containing 10% fetalbovine serum, 1% antibiotics/antimycotic, 0.5% beta-mercaptoethanol, 1%non-essential amino-acids, 1 mM sodium pyruvate, and 10 mM HEPES buffer(all from (Life Technologies, NY, USA)).

Human Primary T Cells and Monocyte Derived Dendritic Cells

Human PBMCs were separated using known methods, e.g., Ficoll-Paque (GEHealthcare, Uppsala, Sweden) density gradient centrifugation from wholeblood. CD4+ T cells were separated from the CD14-negative fraction ofPBMCs using CD14 and CD4 magnetic microbeads (MACS Miltenyi Biotec,Auburn, Calif.). T cells were cultured in RPMI 1640 media (Cellgro,Manassas, Va.) containing 10% Human Serum (AB) (GemCell, WestSacramento, Calif.), 100 U/ml penicillin and streptomycin sulfate 100μg/ml (H 10 medium) supplemented with 5 ng/ml rhIL-15 (R&D Systems,Minneapolis, Minn.) to maintain cell viability without cell activation.Human Monocyte derived Dendritic Cells (MDDCs) were prepared fromCD14-positive monocytes selected from peripheral blood mononuclear cellsusing anti-CD14 magnetic microbeads (MACS Miltenyi Biotec) and culturedfor 6 days with 100 ng/ml interleukin-4 and 50 ng/mlgranulocyte-macrophage colony-stimulating factor (R & D Systems).

Cell Transfection

Human CD45 siRNA: sense (SEQ ID NO: 1)5′-AF488 CUGGCUGAAUUUCAGAGCAdTdT-3′, Human CD4 siRNA: sense(SEQ ID NO: 2) 5′-GAUCAAGAGACUCCUCAGUdTdT-3′ (Alnylam, Cambridge, MA);vif siRNA: sense (SEQ ID NO: 3) 5′-CAGAUGGCAGGUGAUGAUUGT-3′, gag siRNA:sense (SEQ ID NO: 4) 5′-GAUUGUACUGAGAGACAGGCU-3′ (Huang et al., 2013,Nature Biotechnology 31: 350-356); (GenePharma, Shanghai, China);control scrambled siRNA: (SEQ ID NO: 5) 5′-GCCAAGCACCGAAGUAAAUUU-3′,Human DC-SIGN siRNA: sense (SEQ ID NO: 6) 5′-GGAACUGGCACGACUCCAUUU-3′(Dharmacon, ThermoScientific, Pittsburgh, PA).Nucleofection

In the described electroporation experiments, Amaxa Nucleofector II(Lonza Inc., Allendale, N.J.) was used according to the manufacturer'srecommendations. Human T cell experiments were conducted using theprogram for human unstimulated T cells, high viability, U-014 with ahuman T cell kit. For human MDDCs we used the program for humandendritic cells U-002 with a human dendritic cells kit for MDDCs.Briefly 2×10⁶ cells were suspended in 100 μl of Nucleofection solutionwith 200 pmol of siRNA and nucleofected by the machine. To test proteindelivery, we used an APC-labeled mouse IgG1 (cl. MOPC-21, Biolegend) at0.02 mg/ml for both CellSqueeze and nucleofection experiments. We alsoused 3 kDa Cascade Blue labeled dextran and 70 kD Fluorescein labeleddextran at 0.2 mg/ml (Invitrogen).

Regulatory T Cells

Regulatory T cells (Tregs) were isolated and expanded using knownmethods. For example, CD4+ T Cell-enriched PBMC were isolated fromperipheral blood of healthy individuals by density centrifugation usingthe CD4+ T cells RosetteSep enrichment kit (Sigma-Aldrich and STEMCELLTechnologies) and labeled with anti-CD3-PE-Cy7, CD4-FITC, CD25-APC andCD127-PE. CD3+CD4+CD25+CD127low Tregs were sorted on a FACS Aria cellsorter (BD Biosciences), stimulated with anti-CD3/anti-CD28-coatedmicrobeads (Invitrogen) and cultured with IL-2 (300 U/ml).

For siRNA delivery, at day 7 of culture, Tregs were washed andresuspended at 1.0×107 cells/ml in X-VIVO 15 (Lonza) media alone.1.0×10⁶ cells were used per condition. CD4 siRNA(5′-GAUCAAGAGACUCCUCAGU-3′ (SEQ ID NO: 7), Alnylam) and control siRNA(siGENOME Non-Targeting siRNA Pool #1, Thermo Fisher) were used at liAMwith 30-4 chips design at 100 psi.

2 days after siRNA delivery, cells were stained with LIVE/DEAD® FixableViolet Dead Cell Stain Kit (Life Technologies) and anti-CD4-APC. Datawere acquired on a LSR2 flow cytometer (BD Biosciences) and analyzed onFlowJo (Treestar).

Flow Cytometry

Mouse cells were stained with the following antibodies: anti-CD8-PacificBlue, anti-CD4-APC, anti-CD11b-PE (cl. M1/70), anti-CD11c-APC. Propidiumiodide was used to exclude dead cells. Data was acquired using a FACSCantoII, LSR II, or LSRFortessa (BD Biosciences) and analyzed usingFlowJo (Tree Star, Ashland, Oreg.).

Human cells were stained with the following antibodies: anti-CD3-APC(c1.0KT3), anti-CD45RA-PE-Cy7 (cl. HI100) and anti-CD4-AF488 (c1.0KT4)from Biolegend (San Diego, Calif.) and an anti-DC-SIGN-APC (c1.9E9A8) (R& D Systems, Minneapolis, Minn.). Dead cells were excluded using Sytoxblue and 7-AAD (7-Aminoactinomycin D) dead stain dye (Invitrogen). Datawere acquired using a FACS CantoII (BD Biosciences) and analyzed usingFlowJo (Tree Star, Ashland, Oreg.).

HIV Infection and Intracellular p24 Antigen Staining

Primary CD4+ T cells were treated with 5 μM siRNA using a 10-4 chip. Forknockdown of CD4, siRNA was delivered 48 hrs prior to infection whilesiRNA targeting viral genes vif and gag were delivered 24 hrs prior toinfection. The cells were then stimulated overnight with 5pg/mlPhytohaemagglutinin (PHA) and infected with HIVIIIB in 96 wellplates at 2×10⁵ cells/well with HIV IIIB (400 ng/mi p24). HIV IIIB wasobtained from the N1H AIDS Reagent Program and viral stock was preparedas previously described (18). The infection was enhanced by the additionof polybrene at 5 μg/ml and spinoculation at 1200×g, for 2 hrs at 37° C.(19). Intracellular p24 antigen staining was performed 24 hrs laterusing an anti-p24 KC57-FITC Antibody (Beckman Coulter, Fullerton,Calif.) with Fix & Penn Kit for Cell penneabilization (Invitrogen) andanalyzed by flow cytometry.

Quantitative RT-PCR

Total RNA was isolated from T cells using RNeasy Mini Kit (Qiagen) andcopy DNA was synthesized using Superscript III and random hexamers(Invitrogen). Real Time PCR was performed using SsoFast EvaGreen Supemixand a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories,Hercules, Calif.). The primers were as follows: Gapdh forward:5′-AGCCACATCGCTCAGACAC-3′ (SEQ ID NO: 8), Gapdh reverse:5′-GCCCAATACGACCAAATCC-3′ (SEQ ID NO: 9), CD4 forward:5′-GGCAGTGTCTGCTGAGTGAC-3′ (SEQ ID NO: 10), CD4 reverse:5′-GACCATGTGGGCAGAACCT-3′ (SEQ ID NO: 11).

Statistical Analysis

One-way analysis of variance (ANOVA) with Bonferroni's Multiplecomparison test was performed when comparing multiple groups, ortwo-tailed Student's T test was performed when comparing 2 groups usingGraphPad Prism 4 software (GraphPad Software, San Diego, Calif.). *, **and *** indicate P values below 0.05, 0.01 and 0.001 when usingBonferroni's Multiple comparison test, and ### indicate P values below0.001 when using two-tailed Student's T test. Data are represented asmean±1 standard deviation unless otherwise indicated.

Delivery by Mechanical Membrane Disruption

The microfluidic devices tested contained 45-75 parallel microfluidicchannels of varying constriction lengths (10-50 μm), widths (4-9 μm) andnumber of constrictions per channel (1-5 constrictions). (Sharei et al.,2013, Proceedings of the National Academy of Sciences of the UnitedStates of America 110: 2082-2087; Sharei et al., 2014, Integrativebiology: quantitative biosciences from nano to macro 6: 470-475).

The system required to operate the microfluidic chip included a mountingcomponent that secures fluid reservoirs to the silicon and glass device,and a pressure regulation system that controls the gas pressure used todrive the fluid through the system. In some embodiments, the devicecomprises a syringe or pressure source to induce flow through amicrofluidic channel.

The operating procedure is illustrated in FIG. 1A. As cells flow throughthe microfluidic channels (FIG. 1B), they reach a constriction point inthe channel (about 50% less than the diameter of the majority of cellsto be treated) which results in rapid mechanical deformation (exemplarydimensions of constriction point for target cells are as follows T cells(resting: 7-8 μm, activated (7-15 μm), macrophage (resting andactivated: 10-30 μm), dendritic cells (resting and activated: 10-30 μm)of the cell, or squeezing. When the channel constriction isappropriately sized, the deformation transiently disrupts the cellmembrane (e.g., the deformation process takes 0.1 μs-1 ms but themembrane disruptions can stay open for up to 5 min). Macromoleculespresent in the surrounding buffer then enter the cell cytosol if theyare small enough to transit through the membrane disruptions. Within ˜5min, the membrane recovers its integrity and the macromolecules taken upby the cell remain trapped in the cell cytosol. Previous studiesidentified constriction length (L), width (W) and fluid speed (V, notethat fluid speed is determined by operating pressure) as importantparameters that influence delivery efficiency and cell viability.(Sharei et al., 2013, Proceedings of the National Academy of Sciences ofthe United States of America 110: 2082-2087).

A library of 16 different constriction designs were tested underdifferent flow conditions. Library of tested device designs. The firstnumber indicates constriction length, subsequent numbers preceded by adash indicate the width of a constriction. If there are multipleidentical constrictions in series it is indicated by an ‘x’ followed bythe number of constrictions. For example, 10-5-4-5 contains 3 10 μm longconstrictions in series with widths of 5 μm, 4 μm, and 5 μm. 10-4×5contains 5 10 μm long constrictions in series, each with a 4 μm width.

Tested Library of constriction designs 10-4 10-6-4-6 10-6 10-7x5 10-4x230-4 30-6 10-7 10-4x5 30-5-4-5 50-6 10-8 10-5-4-5 30-5x5 10-6x5 10-9Each of the numbers in the width designation “5-4-5” (after the lengthdesignation “10”) representing three different constriction points

These variables included changes in pressure (to change the flow rate)and temperature to optimize macromolecule delivery and minimize cellulartoxicity. All the buffers tested (PBS, PBS+2% serum, complete culturemedia, and whole human blood) were found to be compatible with thesystem, indicating that any physiologically compatible fluid or solutionis suitable for suspending cells through the delivery device andprocess.

30-5×5, 10-4×2, 10-5-4-5, 10-6-4-6, 30-5-4-5, and 10-4×5 designs werealso tested for murine and human T cells, but none was superior to theperformance of 30-4.

TABLE 2 Delivery parameters and their influence on performanceConstriction Constriction geometry (specifically length and width)Design and number of constrictions in series affect delivery efficiencyand cell viability. Longer, narrower, and more numerous constrictionstypically result in more effective delivery but can lead to lowerviability Operating The operating pressure of the system determines thePressure speed at which cells move through the channels and aredeformed. Higher speeds lead to more rapid deformation, which can resultin higher delivery efficiency and potentially lower viability. FlowBuffer The buffer in which cells are suspended during treatment canaffect cell health and may potentially interact with the biomoleculebeing delivered (e.g. serum proteins may bind certain materials). Oneknown effect on delivery is mediated by calcium. The presence of calciumions in the running buffer speeds up membrane repair post-treatment.Operating in calcium- free buffers can increase delivery at the risk ofreducing viability. Operating Lower temperatures (i.e. on ice) improvedelivery Temperature efficiency. Temperature could influence manyparameters. One possibility is that low temperatures retard membranerepair post-treatment.

In some embodiments, the device comprises a constriction length of about5 μm to about 50 μm or any length or range of lengths therebetween. Forexample, the constriction length ranges from about 5 μm to about 40 μm,about 5 μm to about 30 μm, about 5 μm to about 20 μm, or about 5 μm toabout 10 μm. In some embodiments, the constriction length ranges fromabout 10 μm to about 50 μm, about 20 μm to about 50 μm, about 30 μm toabout 50 μm, or about 40 μm to about 50 μm. In some embodiments, theconstriction depth ranges from about gum to about 200 um or any depth orrange of depths therebetween. For example, the constriction depth rangesfrom about 2 μm to about 150 μm, about 2 μm to about 100 μm, about 2 μmto about 50 μm, about 2 μm to about 25 μm, about 2 μm to about 15 μm, orabout 2 μm to about 10 μm. In some embodiments, the constriction depthranges from about 10 μm to about 200 μm, about 25 μm to about 200 μm,about 50 μm to about 200 μm, about 100 μm to about 200 μm, or about 150μm to about 200 μm. In some embodiments, the angle of the entrance orexit portion of the constriction ranges from about 0 degrees to about 90degrees or any angle or range of angles therebetween. For example, theangle is about 5, about 10, about 15, about 20, about 30, about 40,about 50, about 60, about 70, about 80, or about 90 degrees or more. Insome embodiments, the pressure ranges from about 50 psi to about 200 psior any pressure or range of pressures therebetween. For example, thepressure ranges from about 50 psi to about 150 psi, about 50 psi toabout 125 psi, about 50 psi to about 100 psi, or about 50 psi to about75 psi. In some embodiments, the pressure ranges from about 75 psi toabout 200 psi, about 100 psi to about 200 psi, about 125 psi to about200 psi, about 150 psi to about 200 psi, or about 175 psi to about 200psi. In some embodiments, the device comprises a constriction width ofbetween about 2 μm and about 10 μm or any width or range of widthstherebetween. For example, the constriction width can be any one ofabout 3 μm, about 4 μm, about 5 μm, about 6 μm, or about 7 μm.

Delivery to Primary Mouse Cells

To assess the potential of this platform to enable intracellulardelivery to primary immune cells, mouse T cells, B cells, andmonocytes/macrophages were passed through microfluidic devices in thepresence of fluorescently labeled dextran (3, and 70, and 2,000 kDa),and antibodies (about 150 kDa). These materials were selected as modelsfor small molecules, polysaccharides, and proteins, respectively. Atleast four device designs and two operating pressures were tested percell type. The initial selection of constriction dimensions was guidedby work in cell lines, primary fibroblasts and embryonic stem cells.(Sharei et al., 2013, Proceedings of the National Academy of Sciences ofthe United States of America 110: 2082¬2087). Specifically, the widthsof the constrictions were selected to be ˜50% of the average diameter ofthe target cells. Delivery using the 30-4 design (i.e. constriction hasa 30 μm length and 4 μm width) was the most effective for lymphocytesand myeloid cells (FIGS. 2A-C).

Delivery of biomolecules (FIGS. 2A-C and FIG. 6) and cell viability(FIG. 7) were measured by flow cytometry 1-2 hours post-treatment. FIGS.2A-C shows representative histograms of antibody delivery (A), deliveryefficiency of 3 kDa dextran, 70 kDa dextran, and antibodies inindependent experiments (B), and representative median intensity data(C). Delivery efficiency (defined as the percentage of live cells withfluorescence above background) and median fluorescence intensity wereused as the primary measures of delivery. 3 kDa dextran was delivered to66.2±13.4%, 67.5±13.9%, and 80.8±3.46% of T cells, B cells, and myeloidcells, respectively. The uptake of the larger 70 kDa dextran and the Abwere less efficient than uptake of the 3 kDa dextran as expected,suggesting that smaller molecules are more efficiently delivered. Cellviability of T cells, but not B cells or myeloid cells, was somewhatimpaired compared to untreated cells. Changes in viability of B cellsand myeloid cells treated with the device were not significantlydifferent from the untreated or no-device cells. Bone-marrow deriveddendritic cells took up dextran and antibody, with limited loss ofviability, using a larger constriction width of 6 μm because of theirlarger size (FIG. 8). Simultaneous delivery of dextran (3 kDa and 70kDa) and antibody showed that the delivery of these molecules wasproportional, i.e. cells that received antibody, also received acomparative amount of dextran molecules (FIG. 9).

Delivery to Human Immune Cells

To examine the applicability of this approach to human immune cells,device designs with constriction widths ranging from 4-6 μm for T cellsand 6-9 μm for monocyte-derived dendritic cells (MDDCs) were tested.Representative histograms of antibody delivery and efficiency data fordifferent biomolecules are shown in FIG. 3A. The most effective designsdelivered 3 kDa dextran to 70%±9% of T cells (4 μm constriction size)and 60%±4.5% of MDDCs (7 μm constriction size) (FIG. 3A). 70 kDa Dextranwas delivered to 71%±11% of T cells and 40%±14% of MDDCs and protein(antibody) was delivered to 65%±15% of T cells and 38%±7% of MDDCs.Delivery of fluorescently labeled siRNA (CD45RA siRNA—Alexa-Fluor-488)yielded similar results (FIGS. 12, 13).

To test protein knockdown, siRNA against human CD4 or CD45RA wasdelivered to blood derived T cells and observed dose-dependent knockdownof protein levels 72 hours post-treatment using 5, 1 and 0.2 μM siRNA,while control siRNA had not significant effect (FIG. 3B, FIGS. 12, 13).CD4 mRNA was also reduced when measured by qRT-PCR 48 hrs post treatment(FIG. 14). The durability of CD4 knock-down in T cells treated with 5 μMCD4 siRNA was also tested. Knockdown lasted ˜10 days (FIG. 14). Thedelivery of siRNAs targeting the dendritic cell marker DC-SIGN alsoresulted in significant, sequence-specific, knockdown of DC-SIGN proteinexpression in MDDCs using different device designs. The level ofknock-down in different device designs correlated with dextran/Abdelivery efficiency. Representative histograms of protein expression andcompiled knockdown data from independent experiments for CD4 T cells andMDDCs are shown in FIG. 3B. The approach was also found to be applicableto human regulatory T cells (FIG. 3C), B cells and monocytes (FIGS. 16,17, 18).

To compare the performance of the microfluidic device to nucleofection(an electroporation-based approach to nucleic acid delivery) human CD4 Tcells were treated with siRNAs in parallel by the CellSqueeze platformor a nucleofection machine, and protein knockdown was measured 72 hrslater by flow cytometry. Although the extent of CD4 knockdown wassimilar between the two (FIG. 3D), T cell viability post-nucleofection,was significantly worse than in cells treated by the microfluidic device(P<0.05). The efficiency of the two platforms for delivery of labeleddextrans and proteins was compared. Comparison of the performance of thedeformation device to nucleofection, in the context of MDDCs, yieldedsimilar results to T cells. Specifically the best microfluidic device(30-4 for T cells, 10-6 for MDDCs) displayed advantages in cellviability and delivery (FIG. 18). Comparisons of siRNA knockdownefficiency between the two techniques also indicated that cell squeezingcauses less off-target effects (FIG. 19) and improves long-termviability.

Inhibition of HIV-1 Infection in Primary Human T Cells

Studies were carried out to determine whether the microfluidics approachwas useful to inhibit HIV infection and replication in human primaryCD4+ T cells by delivering siRNA targeting viral genes. siRNA againstvif and gag, which were previously shown to suppress viral replication,was delivered to cells 24 hrs prior to infection with HIV. As a positivecontrol, siRNA targeting the HIV receptor CD4 was used. The CD4 siRNAwas delivered 48 hrs prior to infection to ensure reduction of surfaceCD4 levels, thus inhibiting infection. HIV replication was determined bystaining for intracellular p24 antigen and measured by flow cytometry.Representative histograms and compiled results from independentinfection experiments are shown in FIGS. 4A-B. A significant reductionof p24 antigen was observed in T cells treated with vif and gag targetedsiRNA, both separately and in combination (p<0.01). The inhibitoryeffect was greater than that induced by CD4 knock-down (p<0.05) (FIGS.4A-B).

Ex Vivo Cytostolic Delivery of Compounds to Immune Cells Using CellShaped Alteration Device

Intracellular delivery of macromolecules to immune cells was asignificant challenge prior to the invention. Results shown heredemonstrate the utility of a vector-free membrane disruption techniqueto deliver small molecules as well as large macromolecules to eukaryoticcells such as mouse and human immune cells. The results are surprisingand particularly relevant to clinical use, because immune cells arerecalcitrant to other techniques of delivering compositions to thecytoplasm of these cells. The data described herein demonstrates: (i)the ability to deliver a diversity of biologically relevantmacromolecules (polysaccharides, proteins, and nucleic acids); (ii)efficacy in the most clinically-relevant immune cell subsets (T cells, Bcells, DCs, monocytes/macrophages) (FIGS. 2A-C and FIGS. 3A-D); (iii)independence from vector material and electrical fields, thus overcomingsome of the challenges associated with endocytic entrapment andelectroporation-level toxicity; and (iv) the simultaneous delivery ofmultiple classes of macromolecules to target cells (FIGS. 2C, 9, 12,13).

These surprising and significant advantages enable previously unforeseenimmune cell manipulations and clinical applications. For example, thissystem serves as a platform for delivery of peptide or protein-basedtherapeutics to lymphocytes, materials in which existing methods, e.g.electroporation, have shown limited efficacy. The simultaneous deliveryfeature of the system is also useful to screen multiple therapeuticcandidates in parallel to accelerate the screening process andpotentially identify complementary effects. Additionally, the use ofsimilarly sized, labeled, molecules as tracers allows one to monitordelivery efficiency of an unlabeled target material independently. Otherimaging agents, such as quantum dots, may also be delivered using thissystem, thus facilitating direct observation of molecular interactionsin live cells to gain a deeper understanding of biological processes.

In a non-limiting example, a quantum dot or a magnetic nanoparticle isdelivered to facilitate in vivo imaging of transferred immune cells. Forexample, an MM could detect localization of adoptively transferred Tcells loaded with magnetic particles.

The dependence of delivery performance on system parameters, such asconstriction geometry, temperature, operating pressure and buffercomposition are tailored for delivery of compounds/compositions toimmune cells or mixtures of immune cells, e.g., cells in whole blood.For example, exemplary device parameters for immune cells include (Tcell, 30-4 resting, 10-4 activated), B cell (30-4 resting, 10-4activated), macrophage (10-6), dendritic cell (10-6) as well as mixturesuch as white cell fraction/huffy coat cells or whole blood cells. Therange of constriction designs for all is 2-10 μm width, 0.1-90 μmlength). Thus, one could optimize performance of the platform in targetlymphocyte and myeloid cell populations by developing devicearchitectures and operating protocols using design rules established forother cell types. For example, one could increase delivery efficiency byfabricating devices with longer, narrower constrictions. Throughput ofthe system, currently at 100,000-1,000,000 cells/second, can also beincreased by including more parallel channels per device or increasingoperating pressure. In some embodiments, channel depth is increased oradditional channels are added in parallel to increase throughput.Operating pressure may vary depending on the device's design. In certainembodiments, the pressure is 1 psi to 1000 psi.

The vif and gag knockdown studies demonstrate the potential of thisapproach to alter cell phenotype and influence disease-relevantbiological processes, such as viral (e.g., HIV) replication (FIGS.4A,B). This result not only demonstrates the utility of this approachdisease treatment and in studying disease mechanisms (e.g. thedependence of viral replication on specific genes), but alsodemonstrates engineering of immune cells for clinical use by targetingspecific host cell functions/pathways without the use of potentiallytoxic delivery vectors. Protein transcription factors delivered bysqueezing, e.g., IRF5, can be used to increase IFN-α production in humanpDCs, thus demonstrating that proteins delivered by squeezing are alsofunctional and able to influence cell phenotype. This intracellulardelivery system is therefore useful for immune cell engineering withcapabilities beyond that of extracellular antibody and cytokine-basedapproaches.

The data described herein show the surprising efficacy of the viralvector-free, microfluidic approach to cytosolic delivery for immunecells, which (prior to the invention) were difficult to engineer anddifficult to achieve cytosolic delivery of compounds/compositions. Thedata further demonstrates the ability of this approach to facilitate thedelivery of a variety of macromolecules, including polysaccharides,proteins and nucleic acids, to T cells, B cells, myeloid cells (CD11b⁺),and dendritic cells. The functionality of the delivered material wasverified in siRNA-based knockdown studies targeting five differentgenes. Finally, the HIV infection studies underscore the utility of thisapproach to alter cell phenotype and influence viral replication forinhibition of infectious diseases. The vector-free delivery systemdescribed herein provides a safe, reliable, and effective method forengineering the function of immune cells and/or altering theirphenotype, function, state of activation.

Rapid Response Vaccine System for Unidentified Pathogens

Infectious pathogens pose a serious threat to soldiers and civiliansalike. To protect against potential biological attacks and the evolutionof pandemic viruses, one must develop robust, rapid-response vaccinationcapabilities. With state-of-the-art technologies, even if a virulentstrain can be isolated and characterized by scientists, it can takeyears and billions of dollars to develop effective vaccines. Many deadlypathogens, such as Ebola and HIV, still cannot be addressed bycontemporary vaccination methodologies despite decades of study. Theinvention provides methods and devices for rapid-response,multi-targeted, personalized protection against pathogens by directlyengineering an individual's immune cells using the vector-freeintracellular delivery platform described herein. The method enableseffective vaccination of a local at-risk population within hours ofidentifying newly infected individuals and has several advantages overprevious approaches as shown below in Table 3.

TABLE 3 Rapid Attenuated Recombinant Response Vaccine Virus Antigen +Adoptive Vaccination Characteristics Vaccines Adjuvant Transfer PlatformEfficacy High Moderate Moderate/Low High Development Years Months MonthsHours Time (per indication) Development High Moderate High Low/None Cost(per indication) Treatment Cost Low Moderate High Low Field-deploy- YesYes No Yes able

Direct delivery of antigenic proteins to the cytoplasm of anindividual's APCs (e.g., B cells, DCs, and re-programmed T cells)obviates the need for identification and development of attenuated viralvectors while providing more effective protection through inducingmulti-targeted immune responses necessary to counter-act viral escape.The field-deployable vaccination platform addresses an outbreak withinhours, not months/years.

When a pathogen is suspected in an individual, one first takes a biopsythe infected tissue. This tissue, which contains the pathogen, is lysedsuch that the molecular components (i.e. the antigens) remain intact,while, the pathogen is deactivated due to the lysis process. Thisantigen mixture is then delivered to the immune cells of a healthyindividual by drawing their blood and driving their cells through themicrofluidic intracellular delivery device (3), before re-entering theblood stream. This delivery process introduces the pathogen associatedantigens into the cytoplasm of resident APCs in the blood, whichincludes T cells, B cells, dendritic cells and monocytes. The antigenfragments are then presented by the MHC-I and drive the activation ofdisease-specific cytotoxic T cells CTLs to provide protection. Lysis canbe completed within 1-2 hours of obtaining an infected biopsy(generating doses for multiple healthy patients from a single biopsy)and the subsequent inoculation of healthy patients would require <5 minper person. (FIG. 5) Moreover, if a pathogen has been characterized,e.g. in the case of HIV or Ebola, one can substitute the use of celllysate with chemically defined synthetic peptides as the antigen source,thus eliminating potential safety concerns surrounding the use oflysates.

The rapid response vaccine platform provides a first-line defenseagainst a variety of biological threats when a conventional, expensivemulti-year development process can lead to substantial loss of life.This approach to vaccination is also applicable to oncology andinfectious disease treatments.

Microfluidic Squeezing for Intracellular Antigen Loading in B Cells

Antigen presenting cells (APCs) are a diverse subset of immune cells(including dendritic cells, macrophages, B-cells) that capture foreignor self proteins and peptides from tissues, and activate adaptive immunecells to generate either an inflammatory or tolerogenic immune responseagainst these antigens. Proteins are ingested by APCs in vivo viafluid-phase sampling of their surroundings or receptor-mediatedingestion of foreign microbes or dead cell debris. Ingested proteins aredegraded into peptide fragments (antigens) which are processed andpresented to T-cells together with costimulatory signals, instructingnaïve T-cell activation based on the specific signals received by theAPC and the antigens presented. Because of this critical role in T-cellactivation, purified APCs loaded with antigen and activated ex vivo canbe used to expand functional T-cells in culture (e.g., for adoptiveT-cell therapy) or as effective cellular vaccines in vivo. Using themicrofluidic system, ex vivo manipulation of APCs was shown to beeffective as an alternative approach for generating specific types ofimmunity, particularly cytotoxic T lymphocytes (CTLs) in diseases suchas cancer and HIV, where targeted killing of pathogenic cells iscritical and endogenous APC function is actively suppressed. Despitepromising preclinical studies, clinical translation of cell-basedvaccines has been hampered by multiple limitations. The microfluidicdevice and associated methods for delivery of antigen to the cytosol ofimmune cells solves many problems and drawbacks of earlier approaches.

Previous clinical research on cell-based vaccines has focused ondendritic cells (DCs), the so-called—professional—APCs because of theirefficiency in priming CTLs, and their highly active extracellularprotein uptake and antigen-processing capability. However, as a platformfor clinical use, DCs are limited by their relative paucity in humanblood, complex subset heterogeneity, short lifespan, and inability toproliferate. These challenges have led other cell types to also beconsidered for cell-based APC vaccines, including macrophages andB-cells. B-cells are a desirable population of cells for this purpose,because of their unique properties as lymphocytes and their potential toovercome many limitations of DCs. For example, B-cells are abundant incirculation (up to 0.5 million cells per mL of blood), can proliferateupon cellular activation, and efficiently home to secondary lymphoidorgans when administered intravenously.

These advantages of B-cells as APCs are offset by limitations in theability of B-cells to acquire and process antigen for priming ofT-cells. B-cells express genetically rearranged B-cell receptors (BCR),which on binding to their target antigen, promote antigen uptake andB-cell activation. While B-cells are able to internalize antigens viatheir BCRs and prime primary T-cell responses, their uptake ofnon-specific antigens (i.e. antigens not recognized by their BCR) ispoor compared to macrophages and DCs, which efficiently pinocytose andphagocytose antigens from their surroundings. Furthermore, priming ofCTLs occurs through presentation of peptide by class I MHC molecules,which are normally only loaded with antigens located in the cytosol(where the class I MHC processing machinery primarily resides). Bycontrast, proteins taken up via the BCR into endolysosomes tend to bedirected to the MHC class II presentation pathway for presentation toCD4+ T-cells. Alternatively, B-cells and other professional APCs canload class I MHC molecules with peptides via cross presentation, aprocess whereby class I peptide-MHC complexes are produced fromendocytosed antigens via proteasomal processing or vacuolar proteindegradation, but this process is generally very inefficient.

Although methods have been developed to increase antigen uptake andcross-presentation in B-cells, these strategies largely rely ontargeting specific receptors for endocytic uptake, activating B-cellscombined with fluid-phase protein exposure to increase non-specificendocytosis, delivering antigen as immune-stimulating complexes, orgenerating fusion proteins to direct B-cell function. These approachesare limited by the fact that antigen uptake is coupled to other changesin B-cell state mediated by signalling through the targeted receptor,meaning that antigen loading and B-cell activation cannot be separatelytuned. For example, resting B-cells have been shown to be tolerogenic tonaïve CD8+ T-cells, a potentially useful property in treatingautoimmunity, and activation of the B-cell would be problematic in suchan application. Transfection of B-cells with DNA, RNA33, or viralvectors encoding antigens has also shown promise, but has been limitedby a host of issues such as toxicity of electroporation, viral vectorpackaging capacity, transduction efficiency, stability, and anti-vectorimmunity. The methods described herein provide a solution to thesedrawbacks of earlier approaches.

Direct cytosolic delivery of whole proteins, i.e., unprocessed antigen,into live B-cells by transient plasma membrane disruption/perturbation,is accomplished as B-cells are passed through constrictions inmicroscale channels of a microfluidic device (mechano-disruption. Usingthe well-defined and art-recognized model antigen ovalbumin (OVA),delivery of whole protein via this method enabled even resting B-cellsto elicit robust priming of effector CTLs both in vitro and in vivo.This method for whole protein delivery and antigen presentation by MHCclass I is the first antigen delivery method in B-cells that decouplesantigen loading from the process of B-cell activation, allowing thesetwo processes to be separately tailored for immunogenic or tolerogenicvaccines. Cell squeezing provides an alternative modular platform thatprimes autologous B-cells for in vitro CTL expansion as well asfacilitate the development of B-cell-based vaccines.

The following materials and methods were used to generate datapertaining to antigen presentation.

Reagents. TRITC- and Cascade Blue-labelled 3 kDa dextrans were purchasedfrom Life Technologies. FITC-labelled 40 kDa dextran was purchased fromChondrex. Model antigen, Low endotoxin ovalbumin protein was purchasedfrom Worthington Biochemical Corporation. CpG ODN 1826 (CpG B), CpG ODN2395 (CpG C), and LPS Escherichia coli K12 (LPS) were all purchased fromInvivogen. Multimeric/megaCD40L was purchased from Adipogen and EnzoLife Sciences.

Cell isolation. Methods and procedures for isolating/purifying orenriching for immune cells or subsets of immune cells are well known inthe art. For example for humans, peripheral blood mononuclear cells areobtained from whole blood taken by venipuncture and subsets of immunecells, e.g., B cells, T cells, dendritic cells, macrophages, separatedusing standard protocols. Bone marrow is also used as a source of immunecells.

For B-cell isolation in the examples described herein, spleens wereharvested from mice and mashed through a 70 μm cell strainer. Red bloodcells were lysed and resting B-cells were isolated from the cellsuspension with the B-Cell Isolation Kit, mouse (Miltenyi Biotec)following the manufacturer's instructions. After isolation, B-cellsuspensions were >95% B220+, as measured by flow cytometry. ForCD8+T-cell isolation, spleens and inguinal lymph nodes were harvestedand mashed. Following red blood cell lysis, CD8a+T-cells were isolatedwith the CD8a+T-cell Isolation Kit, mouse (Millenyi Biotec) according tothe manufacturer's instructions. CD4+T-cell isolation was performed withthe CD4+T-cell Isolation Kit, mouse (Millenyi Biotec) on spleen andinguinal lymph node suspensions. T-cells were consistently >90% pure, asmeasured by CD8a or CD4 staining and flow cytometry. All cell culturewas performed in T-cell media (RPMI with 10% FBS,penicillin-streptomycin, 1× sodium pyruvate) supplemented with 1 μL/mLof 55 mM β-mercaptoethanol.

Protein delivery by cell squeezing. Delivery of whole protein antigen toresting B-cells was performed using CellSqueeze, a microfluidics deviceand pressure system (SQZ Biotech); chip designs used included 30-4×1,10-4×1, and 30-5×5 where X-Y×Z denotes Z sequential constrictionchannels of dimensions X μm long and Y μm diameter. B-cells weresuspended at 5×106 cells/mL in media with 100 μg/mL of ovalbumin, 0.3mg/mL TRITC- or Pacific Blue-labelled 3 kDa dextran, or 0.3 mg/mLFITC-labelled 40 kDa dextran, and placed on ice. The microfluidics chipsand holder set were also placed in an ice water bath until cold. Thecell suspension was sent through the device in 200 μL aliquots at 120psi. Endocytosis control B-cells were prepared identically in mediumwith OVA, but did not go through the microfluidics device. After antigenloading, cells were allowed to rest at room temperature for 5 minutesand washed twice with PBS. To assess delivery efficiency, uptake ofantigens or other delivered was measured by flow cytometry (detection offluorescently-labeled compositions).

In vitro cell culture, activation & proliferation assays. Tocharacterize in vitro activation of mechano-porated B-cells, cells thatwent through the SQZ device and endocytosis control cells were incubatedin a 96-well U-bottom plate at 5×10⁵ cells/mL with 5 μM CpG B, 5 μM CpGC, or 100 ng/mL LPS. Flow cytometry was performed at 24 and 48 hours tomeasure cell-surface levels of CD86, CD40, CD69, MHC class I, and MHCclass II. For in vitro proliferation assays, purified SIINFEKL ovalbuminpeptide—specific OT-I CD8+(MHC class 1 restricted) or OT-II CD4+ T-cells(MHC class II restricted) were suspended at 107 cells/mL and labelledwith CFSE (5 μM, Life Technologies) for 10 min. After one wash, T-cellsand B-cells were plated at a 1:0.8 ratio in 200 μL of T-cell media in a96-well U-bottom plate. CpG B, CpG C, or LPS was added to theappropriate wells, and anti-CD3/CD28 Dynabeads (Life Technologies) wereadded to positive control wells at 1 bead/T-cell. Supernatant collectionfor cytokine analysis and flow cytometry to assess T-cell proliferationwere performed on day 2 and day 4.

In vivo proliferation assay. On day −1, 106 OT-I Thy1.1 CD8+restingT-cells labelled with CFSE (5 μM) were injected retro-orbitally (r.o.)into C57BL/6 mice. The next day (day 0), animals were injected r.o. with1-3 million CD45.1+B-cells that had been loaded with OVA using themicrofluidics SQZ device the previous day and incubated overnight with 5μM CpG B, or were loaded with OVA by mechano-disruption just prior toinjection and not exposed to any TLR ligand. Animals were necropsied onday 4 and their spleens, inguinal lymph nodes, and cervical lymph nodeswere harvested. The organs were mashed through a cell strainer.Single-cell suspensions were incubated with mouse anti-CD16/CD32(eBioscience) to reduce nonspecific antibody binding, and were stainedwith anti-CD8-APC, anti-B220-PE-Cy7, anti-CD45.1-PerCP-Cy5.5, andanti-Thy1.1-APC-Cy7. Flow cytometry to determine cell numbers and/orCFSE dilution was performed using known methods.

Mechano-Disruption (Microfluidic Cell Squeezing) Enables Rapid andEfficient Delivery of Macromolecules

The process of mechano-disruption for loading B-cells with antigen wasaccomplished using the device described herein. Live cells were passedthrough parallel microfluidic channels in a silicon device; in eachchannel, 1 or more constrictions create transient pores or perturbationsin the membranes of cells passing through the device. Macromolecularcargos present in the surrounding fluid diffuse into the cell duringthis transient perturbation/membrane disruption, leading tointracellular loading. Mechano-disruption is effective for promotingcytosolic delivery of macromolecules into a wide variety of cell typesincluding primary murine B-cells; efficient delivery was achieved with 5sequential constrictions with dimensions 30 μm length and 5 μm width.Device design can be altered (increased number of parallel constrictionchannels to 75, longer entry region, reversibility, etc.) prompted tocustomize mechano-disruption parameters for protein delivery intoB-cells and subsequent antigen presentation. Pilot optimizationexperiments showed that 30-4×1 microfluidics chips (1 constriction perchannel that is 30 μm long and 4 μm in diameter) run at 120 psi wereeffective chip design for mechano-disruption of murine B-cells with bothefficient delivery and high cell viability at concentrate ions of 5×10⁶B-cells/mL. Other configurations are described herein, e.g., 30-5×5 vs.30-4×1. Cells at this pressure ran through a device at a rate ofapproximately 1 million cells per second. Microfluidic squeezingpromoted greatly enhanced dextran uptake compared to endocytosis, withinternalization increased ˜65-fold and ˜25-fold for 3 and 40 kDadextrans, respectively. This represented delivery to 75-90% of all cellsfor both dextrans; by comparison less than 10% of resting B-cellsendocytosed detectable amounts of cargo. Viability of recovered cellsafter mechano-disruption was ˜95%, similar to endocytosis controls. Thecapacity for the maximum number of cells that can be passed throughthese disposable microfluidic chips ranged from 1-5 million cells perdevice; however, devices were run with multiple aliquots of cells (1million cells per run) until clogged, and the maximum number of cellsrun through each individual device in a given experiment was often insignificant excess of 5 million cells. There was low variability inpercentage of delivered cells from first run to clogging of devices,indicating that intra-device variability was minimal. Deliveryperformance of multiple devices within the same experimental session(inter-device variability) was very consistent. Recognizing that someapplications may require higher numbers of B-cells, the efficacy of themicrofluidic devices at different cell densities. The efficiency ofintracellular dextran delivery was largely independent from cellconcentration up to at least 50×10⁶ cells per mL, indicating potentialfor robust scalability.

A schematic showing B cells treated to function as enhanced antigenpresenting cells is shown in FIG. 30.

Polyclonal B-Cells Squeezed with Whole Protein Expand Antigen-SpecificCD8+ T-Cells that Secrete Effector Cytokines In Vitro

To determine whether mechano-disruption can facilitate protein deliveryto the cytosolic class 1 MHC antigen processing and presentationmachinery, we utilized the optimal conditions described above to deliverthe model protein ovalbumin (OVA) into resting, purified polyclonalB-cells, by passing cells through microfluidic device in the presence ofexcess OVA in the surrounding medium. Squeezed B-cells were thenco-cultured with CFSE-labelled OT-I CD8+ T-cells, which bear atransgenic T-cell receptor specific for the MHC class I-restricted OVApeptide SIINFEKL, in the presence or absence of CpG as aB-cell-activating stimulus. CFSE dilution in OT-I T-cells was analysedby flow cytometry to assess proliferation/expansion of the T-cells inresponse to B-cell-presented antigen. Whole protein delivery to B-cellsby cell squeezing was found to enable robust MHC class I antigenpresentation and antigen-specific CD8+ T-cell priming in vitro. Cellsqueezing as described herein primarily directs antigen to the cytosoland not endosomal compartments where MHC class II loading occurs.Consistent with this, the data indicated that neither resting norCpG-activated mechano-porated B-cells were able to expand OVA-specificOT-II CD4+ T-cells after 4 days of co-culture. The functionality ofB-cell-primed CD8+ T-cell populations was assayed by measuring secretionof effector molecules at days 2 and 4 of co-cultures. B-cells loadedwith antigen by squeezing, whether resting or activated, primed T-cellsto secrete substantial quantities of granzyme B, IFN-γ, and TNF-α, whileB-cells loaded with antigen by endocytosis produced basal levels ofcytokines.

Squeezed B-Cells Prime Antigen-Specific CD8+ T-Cells In Vivo

In addition an in vitro antigen-specific T-cell expansion platform,B-cells processed as described are useful as an alternative to dendriticcells for use as cellular vaccines. To evaluate in vivo performance,CFSE-labelled OT-I CD8+ T-cells expressing Thy1.1 as a congenic markerwere adoptively transferred into recipient mice as reporters of antigenpresentation. One day later, resting B-cells were injected immediatelyafter mechano-disruption-mediated antigen loading, ormechano-disruption-loaded B-cells that were subsequently activated for24 h with CpG in vitro were injected. Resting B-cells loaded withantigen by endocytosis were used as controls, either immediately orafter 24 h of activation with CpG. Four days after B-cell transfer, micewere sacrificed and spleens and inguinal lymph nodes were analysed forOT-I proliferation by flow cytometry. Consistent with in vitro results,mechano-porated B-cells were able to elicit division of adoptivelytransferred OT-I T-cells while endocytosis controls showed only basaldivision. Both CpG-activated and resting squeezed B-cells caused OT-Iproliferation in spleens (˜45% and ˜35% divided of injected OT-I T-cellswith p<0.001 and p=0.001 comparing SQZ vs. endocytosis, respectively).CpG-activated and resting squeezed B-cells also elicited similarlyenhanced OT-I proliferation in lymph nodes compared to endocytosisB-cells (˜40% and ˜35% divided of injected OT-I T-cells by CpG B andresting squeezed B-cells, respectively; p<0.001 comparing SQZ vs.endocytosis for both resting and CpG). Endocytosis controls showed ˜4%baseline division in lymph nodes. These results indicated that B-cellsloaded by microfluidic mechano-disruption are useful in cellularvaccines.

The results were further confirmed by experiments evaluating theactivation status of responding CD8+ T cells (FIG. 29).

B-cells loaded with specific antigens as described here are thereforeuseful as APCs for cell-based vaccines and as autologous reagents forexpansion of antigen-specific T-cells, with significant advantages overdendritic cells, especially with respect to their ready availability inlarge numbers from peripheral blood and their ability to be furtherexpanded substantially in culture. Prior to the invention, methods forefficient antigen loading in B-cells, especially for class I MEWpresentation, have been a major barrier to development of polyclonalB-cells as APCs for in vitro or in vivo use. The devices and methodsdescribed herein represent a simple approach using microfluidic-basedmechanical deformation and passive diffusion leading to robust loadingof protein antigens directly to the cytosol of resting or activatedB-cells and to MHC class I presentation of peptides derived from nativewhole protein. The advantages conferred by mechano-disruption arenumerous, including delivery of native proteins without processing,engineering, or modification, the rapid nature of the process, therelatively high efficiency of delivery and functional outcomes, and theability to deliver material to resting cells decoupled from cellularbiology such as programming stimulus or receptor targeting.

The ability to deliver whole native protein or polypeptides directly tothe cytosol for processing and MHC class I presentation enables unbiasedpresentation of multiple peptides following native antigen processing.The microfluidic cell squeezing device also enables delivery of mixturesof proteins, including tumor lysates, e.g., tumor biopsy lysates, orother complex protein sources. The foregoing demonstration that proteinswere delivered for MHC class I processing independently of cellactivation status is a major advantage of this approach, and theapproach enables decoupling of protein loading and cellular programming,facilitating independent modulation of each component.

An exemplary design contains 75 parallel constriction channels; however,increases in the number of channels or operating multiple devices inparallel dramatically improves throughput. The ease of use and rapidprocessing enabled by this approach (e.g., ˜1×10⁶ cells/s rate flowthrough device, <2 h for total experiment time) provides benefits forclinical translation such as reduction in time and resources requiredfor preparation of cell-based therapeutics.

Using B-cells as APCs, the amount of patient blood required to prepare asingle-dose cellular vaccine is vastly reduced compared to DC-basedapproaches. Time required for protein loading by cellular uptakeprocesses such as endocytosis or pinocytosis is avoided. The results invitro demonstrated significant potential for B-APCs as an alternativeplatform for expansion of effector CTLs and the squeezed B-cells alsofunctioned as effective APCs in vivo. The methods are also useful toco-load both MHC class I and class II antigen presentation pathways togenerate CD4+ T-cell help.

Class I-Restricted Antigen Processing and Presentation by B Cells

Thus, B-cells were processed by cell squeezing to yield autologousantigen-presenting cells (APCs) to prime antigen-specific T-cells bothin vitro and in vivo. This microscale cell squeezing process createstransient perturbations or pores in the plasma membrane, enablingintracellular delivery of whole proteins from the surrounding mediuminto B-cells via mechano-disruption. Both resting and activated B-cellsprocess and present antigens delivered via mechano-disruptionexclusively to antigen-specific CD8+ T-cells, and not CD4+ T-cells.Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolyticfunction, including granzyme B and interferon-γ. The squeezedantigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. These datademonstrate that mechano-disruption/membrane perturbation is a usefuland highly efficient method for B-cell antigen loading, priming ofantigen-specific CD8+ T-cells, and decoupling of antigen uptake fromB-cell activation.

T Cells as Antigen Presenting Cells for Immune Stimulation or Tolerance

Prior to the invention, antigen presentation for the purposes of immunestimulation or tolerance was generally assumed to be the purview of aselect subset of cells referred to as antigen presenting cells, e.g., Bcells, dendritic cells and macrophages, and did not include T cells,because they were assumed to lack the necessary machinery to process andpresent antigens in a context that would facilitate immune stimulationor tolerance. The devices and squeeze-mediated antigen loading of cellsled to a surprising discovery that delivering antigenic proteinsdirectly to the cytosol of T cells confers onto such processed cells anantigen presentation phenotype.

The methods modulate T cell immune responses in vivo in a manner thathad not been reported previously. A model antigen, ovalbumin, wasdelivered to murine primary T cells using the Cellsqueeze deviceplatform, and the cells were injected into hosts to measure CD8+ T cellresponses. The antigen loaded T cells were capable of generating asubstantial CD8+ T cell responses as measured by flow cytometry,indicating that T cells loaded with antigens by an external mechanism,e.g., cell squeezing, can indeed present epitopes on their surface,communicate with other T cells, and generate a measurable immuneresponse. This response was significantly greater than controls and wascomparable to responses observed in experiments using professional APCssuch as dendritic cells.

This finding was unexpected and is of great significance to the field ofimmunology and immunotherapy as it demonstrates the use of T cells aseffective antigen presenters for therapeutic and research applications.Prior to the invention, DCs were the only cell widely accepted for thisuse, and as is described above, the cell-squeeze approach is useful forrapid, efficient, and greatly-enhance production of B cells for thispurpose. Because T cells are more abundant and accessible than DCs, theyprovide greater clinical efficacy and facilitate broader impact forcurrently expensive, difficult to produce cellular vaccine applications.Thus, a T cell can now be used as an antigen presenting cell forpresentation for any type of antigen, purpose (tolerogenic vs.immunostimulatory), routes of injection, for clinical as well asresearch applications.

Mouse T cells treated with whole, unprocessed antigen using thecell-squeeze method function as antigen presenting cells (FIG. 29).Murine T cells were isolated from mouse spleens and OVA was delivered tothe T cells in RPMI at OVA concentrations of (250, 50, 10 ug/ml OVA)using the exemplary 10-3 and 30-4 device configurations. These T cellswere then cultured with OT-1 T cells (SIINFEKL peptide epitope-specific)and activation markers CD25 and CD69 were assessed. The resultsdemonstrate that T cells into which whole, unprocessed antigen, e.g.,full-length ovalbumin, was cytosolically delivered using cell-squeezingsurprisingly and effective function to present antigen to and activateepitope-specific effector CD8+ T cells.

Additional data demonstrate the delivery of DQ-Ova (DQ™ ovalbumin,Catalog # D-12053, Molecular Probes, Inc.) to primary human T cellsisolated from blood (FIGS. 28A and B). The DQ ova is a chemicallyconjugated version of ovalbumin protein that fluoresces on the FITCchannel if the protein is processed but will not fluoresce if theprotein was not processed by the cell. Thus, the appearance of a FITCsignal for the device-treated cases indicates that the cells wereprocessed the DQ-Ova antigen, further supporting the observation thatthe manufacturing antigen-presenting T cells (T cells as APCs) functionin a human system in addition to mouse system described above. In someexperiments, 3 kDa Dextran dye that fluoresces on the pacific bluechannel was co-delivered. Results show differences in Ova processingbetween CD4 and CD8 T cells and a dramatic improvement relative to theendocytosis control. These data indicate that antigen processing iscarried out by human T cells when the antigen is delivered directly tothe cytosol of the Tcell. Data showing that the DQ¬Ova antigen changesits fluorescence properties show that the antigen is being processed inhuman T cells and therefore presented by histocompatibility antigens toT effector cells.

Activation of epitope-specific T cells was also demonstrated in vivo.FIG. 26 shows the results of a CFSE proliferation assay in vivo (theproliferation of antigen specific CD8+ T cells). As the CD8T cells areactivated and proliferate in the mouse, they dilute the CFSE dye andhave a lower fluorescence intensity. In this case, donor T cells treatedby the device or the positive control yielded significantly greateractivation and proliferation of CD8 T cells in the recipient mouse.Endocytosis controls by contrast show minimal effect.

Proliferation of antigen specific OT-I T cells in mice in response tovaccination with antigen treated wild type T cells was also demonstratedin vivo (FIG. 27). T cell proliferation responses were measured by CFSEstaining. The stain is diluted as the cells proliferate; thus, lowerintensities indicate greater responses, higher intensity peaks indicateno/less response. Each column represents a replicate of the experimentwith the lymph nodes and spleen derived from the same mouse. Each columnof experiments involved 3 mice (n=9 mice in total).

The materials and methods described below were used to generate datapertaining to T cells as Antigen Presenting Cells for Immune Stimulationor Tolerance described above. Experimental Timeline. The followingexperimental timeline was used:

Day 0: Inject CFSE/CellTraceViolet labeled OT-1 CD45.1 T cells intonaïve B6 hosts

Day 1: Deliver antigen to T cell APCS (Tc-APCs), inject Tc-APCs andcontrol APCs into B6 hosts

Day 5/6: Harvest spleens and lymph nodes from immunized mice, analyze Tcell proliferation via flow cytometry

T cell isolation (for adoptive transfer). Materials: 100 urn Falcon cellstrainer; 70 μm Falcon cell strainer; Ammonium-Chloride-Potassium (ACK)lysis buffer; CD8a+ T cell isolation kit (Miltenyi Biotec); MACS cellseparation column. T cell media (RPMI with 10% FBS, pen strep, 1× sodiumpyruvate, 50 uM b-mercaptoethanol); MACS buffer (0.5% BSA, 2 mM EDTA inPBS). Methods: Spleens and skin draining lymph nodes were harvested fromOT-1 CD45.1 Rag 2−/− mice on a C57BL/6J background and mashed through awet 100 μm cell strainer into a 50 mL Falcon tube. Filters were washedwith MACS buffer, then spun down at 500 rcf for 4 minutes. Supernatantswere aspirated, followed by the addition of 3 mL of ACK lysis buffer tolyse red blood cells. Reactions were quenched with 12 mL of MACS buffer,then spun down at 500 ref for 4 minutes. Cell pellet was resuspended in40 uL MACS buffer per 1×10⁷ cells, then filtered through a 70 μm cellstrainer into a 50 mL Falcon tube. 10 uL of CD8a+ T cell antibodycocktail were added per 1×10⁷ cells and incubated for 10 minutes overice. 30 uL MACS buffer per 1×10⁷ cells were added, then 20 uL ofstreptavidin beads were added per 1×10⁷ cells and incubated over ice for15 minutes. Cells were spun down at 800 rcf for 5 minutes andsupernatant was aspirated, then resuspended in 3 mL MACS buffer. Cellseparation column was prepared by passing through 3 mL of MACS buffer,followed by 3 mL of cell suspension over a 70 μm cell strainer, into a15 mL Falcon tube. Column was washed 3× with 3 mL MACS buffer, pooledflowthrough was spun down at 500 rcf for 4 minutes. Supernatant wasaspirated and enriched CD8+ T cells were suspended in T cell media. >90%purity was checked by staining for CD8a and analysis on flow cytometry.

CellTrace Violet/CFSE Labeling. Materials: CFSE in DMSO (LifeTechnologies, Grand Island, N.Y.); CellTrace Violet in DMSO (LifeTechnologies, Grand Island, N.Y.); Purified OT-I CD45.1 CD8+ T cells;Sterile PBS; Sterile fetal bovine serum. Methods: OT-1 CD45.1 CD8+ Tcells were enriched as described above and suspended in 2.5 mL of warmPBS in a 15 mL Falcon tube. CellTrace Violet or CFSE in DMSO wasdissolved in warm PBS at 10 uM, then 2.5 mL was added to cell suspension(5 uM final concentration). Cells were left in a 37° C. warm water bathfor 10 minutes, then 7 mL of PBS was added to quench reaction. 2 mL ofFBS was layered to the bottom using a sterile glass pipette, then spundown at 500 ref for 4 minutes. Supernatent was aspirated and cells werewashed again in 10 mL of PBS, then spun down at 500 rcf and resuspendedat 2×10⁷ cells per 100 ul PBS.

T cell isolation (as Tc-APCs). Materials: 100 um Falcon cell strainer;70 um Falcon cell strainer; ACK lysis buffer; Pan T cell isolation kit(Miltenyi Biotec, San Diego, Calif.); MACS cell separation column; Tcell media (RPMI with 10% FBS, pen-strep, lx sodium pyruvate, 50 uMb-mercaptoethanol); MACS buffer (0.5% BSA, 2 mM EDTA in PBS). Methods:Spleens and skin draining lymph nodes were harvested from C57BL/61 miceand mashed through a wet 100 μm cell strainer into a 50 mL Falcon tube.Filters were washed with MACS buffer, then spun down at 500 rcf for 4minutes. Supernatants were aspirated, followed by the addition of 3 mLof ACK lysis buffer to lyse red blood cells. Reactions were quenchedwith 12 mL of MACS buffer, then spun down at 500 rcf for 4 minutes. Cellpellet was resuspended in 40 uL MACS buffer per 1×10⁷ cells, thenfiltered through a 70 urn cell strainer into a 50 mL Falcon tube. 20 uLof Pan T cell antibody cocktail were added per 1×10⁷ cells and incubatedfor 10 minutes over ice. 30 uL MACS buffer per 1×10⁷ cells were added,then 20 uL of streptavidin beads were added per 1×10⁷ cells andincubated over ice for 15 minutes. Cells were spun down at 800 rcf for 5minutes and supernatant was aspirated, then resuspended in 3 mL MACSbuffer. Cell separation column was prepared by passing through 3 mL ofMACS buffer, followed by 3 mL of cell suspension over a 70 μm cellstrainer, into a 15 mL Falcon tube. Column was washed 3× with 3 mL MACSbuffer, pooled flowthrough was spun down at 500 rcf for 4 minutes.Supernatant was aspirated and enriched T cells were suspended in T cellmedia. >90% purity was checked by staining for CD3e and analysis on flowcytometry.

T cell antigen delivery and activation in vitro. Materials: Purified Tcells; CellSqueeze 30-4 chips; CellSqueeze device; Lipolysaccharide(LPS); T cell media (RPMI with 10% FBS, pen-strep, lx sodium pyruvate,50 uM b-mercaptoethanol); OVA protein; SIINFEKL peptide. Methods:Purified T cells were incubated in T cell media with 1 ug/mL LPS for 30minutes over ice. T cells were washed 2× in T cell media and spun downat 500 rcf for 4 minutes, then incubated with either 0.01 mg/mL-0.1mg/ml OVA or 1 ug/mL SIINFEKL over ice. CellSqueeze apparatus wasassembled according to standard protocol with the 30-4 chip, then 200 uLof T cell media was run through each chip at 100 PSI. Half of the Tcells were passed through CellSqueeze device at 100 PSI and the otherhalf were left over ice (endocytosis case). Flowthrough was collected ina 96 well plate and left over ice for 15 minutes for cell membranes torepair. Cells were spun down at 350 RCF for 10 minutes and supernatantswere flicked. Cells were reactivated in 1 ug/mL LPS for 10 minutes priorto injection into hosts.

Enrichment of Patient Immune Cells

Cell squeezing is also useful to enrich or expand patient-derived immunecells ex vivo for subsequent transfer back into the patient. Forexample, antigen is delivered via mechano-disruption to the cytosol ofantigen presenting cells (dendritic cells, B cells, T cells,macrophages). These antigen presenting cells process and present theprocessed antigen in the context of MHC/HLA heterodimers on theirsurface to stimulate and expand T cells. The expanded populations of Tcells are then re-infused in the patient to augment a therapeutic immuneresponse. For example, the antigen is a tumor antigen (or lysate) toaugment an anti-tumor response. Alternatively, the antigen or abacterial or viral antigen (or fragments thereof or attenuated or killedbacterial cells or viral particles) to augment microbial infectiousdiseases. In another example, the antigen is a self-antigen presentedtogether with a tolerogen (as described) above) to tolerize and thenexpand a population of tolerized immune cells for re-infusion back intothe patient to downregulate an aberrant, e.g., auto-immune, response.

Use of T Cells as Antigen Presenting Cells

Cancer immunotherapy based on the activation of a patient's T cellsusing antibody therapeutics has shown success in those indications withhigh frequencies of mutations and with pre-existing T cell responses totumor associated antigens (TAA)(Topalian et al., Cancer Cell 2015; 27:450-461; Hodi et al., N Engl J Med 2010; 363: 711-723; Topalian et al.,Cell 2015; 161: 185-186). Because many cancers have low frequencies ofmutations and low rates of spontaneous T cell responses, the use ofcancer vaccines may serve to boost T-cell responses to TAA (Melief etal., J Clin Invest 2015; 125: 3401-3412). Cancer vaccines have thepotential to be used as a monotherapy (Ly et al., Cancer Res 2010; 70:8339-8346; Kantoff et al., N Engl J Med 2010; 363: 411-422), incombination with checkpoint blockade therapy (Agarwalla et al. JImmunother 2012; 35: 385-389) or, in combination with adoptive T-celltherapy (Lou et al., Cancer Res 2004; 64: 6783-6790).

In the preclinical setting, strategies using professional antigenpresenting cells (APC) for cancer vaccination have demonstrated thatantigen-pulsed dendritic cells (DC) can generate protective immunityagainst tumors (Celluzzi et al., J Exp Med 1996; 183: 283-287; Mayordomoet al., Nat Med 1995; 1: 1297-1302; 7489412; Flamand et al., Eur JImmunol 1994; 24: 605-610). Clinical studies performed in various typesof cancer (Nestle et al., Nat Med 1998; 4: 328-332; Hu et al., CancerRes 1996; 56: 2479-2483; Hsu et al., Nat Med 1996; 2: 52-58; Reichardtet al., Blood 1999; 93: 2411-2419; Morse et al., Clin Cancer Res 1999;5: 1331-1338; Yu et al., Cancer Res 2001; 61: 842-847) suggest thatmonocyte-derived DCs are capable of eliciting antigen-specific immunityin humans, however the clinical responses are low. For example, thefirst FDA approved DC-based cancer vaccine (Sipuleucel-T, Provenge,Dendreon, Seattle, Wash.) demonstrated four months prolonged survival inpatients with hormone-refractory prostate cancer (Kantoff et al., N EnglJ Med 2010; 363: 411-422; Higano et al., Cancer 2009; 115: 3670-3679).Among the hypotheses to explain why this vaccine has such low clinicalbenefit, the procedure used to load the Sipuleucel-T DCs with antigen,may have resulted primarily in MEW Class II antigen presentation asopposed to having both MHC Class I and II presentation. As such, lowcytotoxic T cell activity may have had a role in the poor efficacy ofthis product.

Methods developed for delivery of TAA to DCs utilize the CellSqueezeplatform for optimal antigen presentation on both MHC Class I and IIhistocompatibility molecules. By addressing the limitations of existingantigen loading techniques, the methods described herein lead to morepowerful cytotoxic and helper T cell responses in vivo. The platformincludes a microfluidic chip capable of rapidly deforming cells as theypass through a constriction to temporarily disrupt their membrane andenable transport of the target material to the cell cytoplasm. Byeliminating the need for electrical fields or exogenous enhancers orcarrier materials, the methods described minimizes the potential forcell toxicity and off-target effects.

Targeting Viral or Neo Antigens

Since central tolerance can be induced to self antigens, therapeuticvaccinations are now preferably targeting neo (Gubin et al., J ClinInvest 2015; 125: 3413-3421; Gubin et al., Nature 2014; 515: 577-581;Yadav et al., Nature 2014; 515: 572-576; Quakkelaar et al., Adv Immunol2012; 114: 77-106; Castle et al., Cancer Res 2012; 72: 1081-1091) andviral antigens (Melief et al., J Clin Invest 2015; 125: 3401-3412;Quakkelaar et al., Adv Immunol 2012; 114: 77-106).

To identify and evaluate antigens, a cocktail of nine synthetic longoverlapping peptides (SLP) of the oncogenes E6 and E7 of HPV16(HPV16-SLP) that represents the entire length of these two oncoproteins,and has shown responses in preclinical models as well as in patientswith premalignant are used to evaluate immune responses. The CellSqueezeplatform is used to load DC with the HPV16-SLP cocktail, which allowspeptides to be channeled into both MHC class I and II presentationpathways, and thus induces antigen-specific expansion of CD4 and CD8 Tcells. As such, this approach prompts a multi-epitope response thatovercomes the limitations of HLA specific short peptides and enablesmore effective therapies.

CellSqueezing of HPV16-SLP to Human and Mouse DC.

Different CellSqueeze conditions are tested to deliver the HPV16-SLPcocktail to human and mouse DC. Chip designs having variations inconstriction length, constriction width, the number of constrictions andthe angle of approach to the constriction are evaluated. With regard tothe process parameters, cell concentration, delivery medium, peptideconcentration in the delivery medium, processing temperature, operatingpressure and the number of passes through the chip are investigated. Theexperimental approach for the optimization process also includesfluorescent labeling of the HPV16-SLP peptide cocktail to account forthe amount of peptide delivery into cells. Moreover, optimal processingand presentation of the peptides is accounted for in the context ofclass I and II by detecting the presented peptides with TCR specifictetramers by flow cytometry. This approach is compared to the loading ofDC with HLA specific Class I short peptides for cross-presentation.

This study identifies chip designs and delivery conditions thatefficiently load human and murine DC with the HPV16-SLP peptide antigensor mixtures of antigens, e.g., a cocktail of viral antigens, e.g.,including conditions for efficient processing of long peptides andloading of antigens by APCs, e.g., dendritic cells.

In Vitro and In Vivo Assessment of the .otenc of HPV 6-SLP squeezed DCto Expand Antigen Specific T Cells.

The function of DC loaded with the peptide cocktail under severalconditions for their ability to expand antigen specific T cells istested. For example, antigen-specific T cells are induced by immunizingmice with HPV16-SLP and CpG. Eight days after boosting, T cells areisolated from spleens of immunized mice and are used to co-culture withHPV16-SLP loaded DC in order to determine the capability of loaded cellsto trigger antigen-specific CD4 and CD8 T cell expansion and cytokineproduction. In order to test the human loaded DC, human T cell clonesreactive to HPV16 are used to co-culture with human DC loaded with theHPV16-SLP cocktail under several conditions. Antigen-specific T cellexpansion and cytokine production are assessed. Whether human and/ormouse loaded DC are functional and capable of expanding antigen-specificT cells is tested in vitro. DC loaded with HLA specific Class I shortpeptides are used as controls. HLA matching T cells are used asresponders.

Thereafter and to test for the effectiveness of the CellSqueeze DC-basedvaccine to generate anti-tumor responses against HPV-16 in vivo, miceare vaccinated with HPV16-SLP loaded DC and challenged with TC1 tumors(retrovirally transduced lung fibroblast of C57BL/6 mice with HPV E6 andE7, and c-H-ras oncogenes). Survival of the challenged mice is thereadout.

These studies show that a DC-based vaccine generated with CellSqueezetechnology triggers protective anti-tumor T cell and/or anti-viralresponses in vivo.

OTHER EMBODIMENTS

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference. While this invention has been particularlyshown and described with references to preferred embodiments thereof, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

The invention claimed is:
 1. A method for engineering of immune cellfunction in an immune cell comprising intracellular delivery of acompound by passing a suspension comprising said immune cell through amicrofluidic device comprising a constriction and contacting the immunecell with said compound to deliver said compound into the cytosol ofsaid immune cell, wherein the constriction has a diameter of 2 μm to 10μm, and wherein said compound: a) comprises a differentiation factor, aself-amplifying RNA that encodes for continual production of an encodedprotein, a chimeric antigen receptor, a nucleic acid encoding a chimericantigen receptor, a tolerogenic factor, an adjuvant, a virus, avirus-like particle, a nucleic acid that is a plasmid, a nucleic acidthat is a transposon, or a nucleic acid that encodes an MHC complex; orb) confers the expression of a homing phenotype to the immune cell; orc) induces the differentiation of the immune cell into a cell with atolerogenic phenotype; or d) enhances immune cell function, wherein theimmune cell is a T cell, and wherein the compound that enhances T cellfunction is an immune checkpoint pathway inhibitor.
 2. The method ofclaim 1, wherein the compound comprises a differentiation factor.
 3. Themethod of claim 1, wherein said membrane is disrupted by passing saidimmune cell through a constriction having a diameter of 3 μm to 6 μm. 4.The method of claim 1, wherein said immune cell is a T cell, B cell,dendritic cell, macrophage, NK cell, monocyte, neutrophil, granulocyte,or innate lymphoid cell.
 5. The method of claim 1, wherein said compoundconfers the expression of a homing phenotype to the immune cell.
 6. Themethod of claim 1, wherein said compound induces the differentiation ofthe immune cell into a cell with a tolerogenic phenotype.
 7. The methodof claim 1 wherein said compound comprises a self-amplifying RNA thatencodes for continual production of an encoded protein.
 8. The method ofclaim 1, further comprising contacting said immune cell with an effectorT cell and activating a cytotoxic T cell immune response.
 9. The methodof claim 1, further comprising contacting said immune cell with aneffector T cell and activating a helper T cell immune response.
 10. Themethod of claim 1, further comprising contacting said immune cell withan effector T cell and activating a tolerogenic T cell immune response.11. The method of claim 1, wherein said compound comprises a chimericantigen receptor.
 12. The method of claim 11, wherein the chimericantigen receptor is a chimeric T cell receptor.
 13. The method of claim1, wherein said compound enhances immune cell function.
 14. The methodof claim 13, wherein the immune cell is a T cell.
 15. The method ofclaim 14, wherein the compound that enhances T cell function is animmune checkpoint pathway inhibitor.
 16. The method of claim 1, whereinsaid compound comprises a tolerogenic factor.
 17. The method of claim 1,wherein said compound comprises an adjuvant.
 18. The method of claim 1,wherein said compound comprises a nucleic acid.
 19. The method of claim18, wherein said nucleic acid is or encodes an siRNA, mRNA, miRNA,lncRNA, tRNA, saRNA, or shRNA.
 20. The method of claim 18, wherein saidnucleic acid is a plasmid.
 21. The method of claim 1, wherein thecompound comprises a protein or peptide.
 22. The method of claim 1,wherein the compound comprises a virus.
 23. The method of claim 18,wherein the nucleic acid encodes an MHC complex.
 24. The method of claim1, wherein said immune cell is in a resting state.
 25. The method ofclaim 24, wherein expression of one or more of the markers CD25, KLRG1,CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7,CX3CR1 or CXCR5 in the immune cell is modulated by delivery of saidcompound into said immune cell.
 26. The method of claim 25, whereinexpression of one or more of the markers is decreased and/or expressionof one or more of the markers is increased.
 27. The method of claim 1,wherein said immune cell is a naive immune cell or a memory immune cell.28. The method of claim 1, wherein said cell suspension comprises wholeblood, buffy coat cells, a mixed cell population, or a purified cellpopulation.
 29. The method of claim 1, wherein said cell suspensioncomprises peripheral blood mononuclear cells, and wherein the compoundis delivered to a peripheral blood mononuclear cell of the cellsuspension.
 30. The method of claim 1, wherein said cell suspensioncomprises mammalian cells.
 31. The method of claim 30, wherein said cellsuspension comprises human, mouse, dog, cat, horse, monkey, or ratcells.
 32. The method of claim 1, wherein said compound comprises anucleic acid encoding a chimeric antigen receptor.
 33. The method ofclaim 32, wherein the chimeric antigen receptor is a chimeric T cellreceptor.
 34. The method of claim 1, wherein said compound comprises avirus-like particle.
 35. The method of claim 1, wherein said nucleicacid is a transposon.
 36. The method of claim 29, wherein the peripheralblood mononuclear cells comprise one or more of the following types ofcells: T cells, B cells, NK cells, monocytes, dendritic cells, andmacrophages.
 37. The method of claim 29, wherein the peripheral bloodmononuclear cells comprise a T cell.
 38. The method of claim 29, whereinthe peripheral blood mononuclear cells comprise a B cell.
 39. The methodof claim 29, wherein the peripheral blood mononuclear cells comprise anNK cell.
 40. The method of claim 29, wherein the peripheral bloodmononuclear cells comprise a monocyte.
 41. The method of claim 29,wherein the peripheral blood mononuclear cells comprise a dendriticcell.
 42. The method of claim 29, wherein the peripheral bloodmononuclear cells comprise a macrophage.
 43. The method of claim 37,wherein the compound comprises a chimeric antigen receptor.
 44. Themethod of claim 37, wherein the compound comprises a nucleic acidencoding a chimeric antigen receptor.
 45. The method of claim 37,wherein the compound comprises a nucleic acid that is a transposon. 46.The method of claim 37, wherein the compound enhances T cell function,and wherein the compound that enhances T cell function is an immunecheckpoint pathway inhibitor.
 47. The method of claim 37, wherein thecompound comprises a virus or virus-like particle.
 48. A method forengineering of immune cell function in an immune cell comprisingintracellular delivery of a compound by passing a suspension comprisingsaid immune cell through a microfluidic device comprising a constrictionand contacting the immune cell with said compound to deliver saidcompound into the cytosol of said immune cell, wherein the constrictionhas a diameter of 2 μm to 10 μm, and wherein said compound comprises: a)an antigen that comprises a full-length, unprocessed protein; or b) atumor antigen or viral antigen selected from the group consisting ofliver cancer antigen, lung cancer antigen, bladder cancer antigen,breast cancer antigen, colon cancer antigen, rectal cancer antigen,endometrial cancer antigen, kidney cancer antigen, leukemia antigen,melanoma antigen, non-Hodgkin lymphoma antigen, pancreatic cancerantigen, prostate cancer antigen, thyroid cancer antigen, ovarian cancerantigen, uterine cancer antigen, HIV antigen, Ebola antigen, HPVantigen, and EBV antigen.
 49. The method of claim 48, wherein saidcompound comprises an antigen having a length of greater than 7, 8, 9 or10 amino acids and wherein said immune cell processes said antigen anddisplays a class I histocompatibility antigen restricted processed formof said antigen on a surface of said immune cell.
 50. The method ofclaim 48, wherein the compound comprises an antigen, and wherein saidimmune cell processes said antigen and displays said antigen on asurface of said immune cell.
 51. The method of claim 50, wherein theimmune cell displays a class I or class II histocompatibility antigenrestricted processed form of said antigen on a surface of said immunecell.
 52. The method of claim 48, wherein said membrane is disrupted bypassing said immune cell through a constriction having a diameter of 3μm to 6 μm.
 53. The method of claim 48, wherein said immune cell is a Tcell, B cell, dendritic cell, macrophage, NK cell, monocyte, neutrophil,granulocyte, or innate lymphoid cell.
 54. The method of claim 48,further comprising intracellular delivery of a compound that induces thedifferentiation of the immune cell into a cell with a tolerogenicphenotype.
 55. The method of claim 48, wherein said antigen comprises afull-length, unprocessed protein.
 56. The method of claim 48, furthercomprising contacting said immune cell with an effector T cell andactivating a cytotoxic T cell immune response.
 57. The method of claim48, further comprising contacting said immune cell with an effector Tcell and activating a helper T cell immune response.
 58. The method ofclaim 48, further comprising contacting said immune cell with aneffector T cell and activating a tolerogenic T cell immune response. 59.The method of claim 48, wherein said compound comprises adisease-associated antigen.
 60. The method of claim 55, wherein saidcompound comprises a tumor antigen, viral antigen, bacterial antigen,self-antigen, or fungal antigen.
 61. The method of claim 48, whereinsaid compound comprises a liver cancer antigen, lung cancer antigen,bladder cancer antigen, breast cancer antigen, colon cancer antigen,rectal cancer antigen, endometrial cancer antigen, kidney cancerantigen, leukemia antigen, melanoma antigen, non-Hodgkin lymphomaantigen, pancreatic cancer antigen, prostate cancer antigen, thyroidcancer antigen, ovarian cancer antigen, or uterine cancer antigen. 62.The method of claim 48, wherein said compound comprises a tumor celllysate or cell lysate from tissue infected with a pathogen.
 63. Themethod of claim 48, wherein said compound comprises a neoantigen. 64.The method of claim 48, wherein said compound comprises an HIV antigen,Ebola antigen, HPV antigen, or EBV antigen.
 65. The method of claim 48,further comprising intracellular delivery of a compound that enhancesimmune cell function.
 66. The method of claim 48, wherein the immunecell is a T cell.
 67. The method of claim 66, further comprisingintracellular delivery of a compound that enhances T cell function. 68.The method of claim 67, wherein the compound that enhances T cellfunction is an immune checkpoint pathway inhibitor.
 69. The method ofclaim 48, further comprising intracellular delivery of a tolerogenicfactor.
 70. The method of claim 48, further comprising intracellulardelivery of an adjuvant.
 71. The method of claim 61, wherein thecompound comprises a protein or peptide.
 72. The method of claim 48,wherein the compound comprises a virus or virus-like particle.
 73. Themethod of claim 48, wherein said immune cell is in a resting state. 74.The method of claim 73, wherein expression of one or more of the markersCD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II,CD62L, CCR7, CX3CR1 or CXCR5 in the immune cell is modulated by deliveryof said compound into said immune cell.
 75. The method of claim 74,wherein expression of one or more of the markers is decreased and/orexpression of one or more of the markers is increased.
 76. The method ofclaim 48, wherein said immune cell is a naive immune cell or a memoryimmune cell.
 77. The method of claim 48, wherein said cell suspensioncomprises whole blood, buffy coat cells, a mixed cell population, or apurified cell population.
 78. The method of claim 48, wherein said cellsuspension comprises peripheral blood mononuclear cells, and wherein thecompound is delivered to a peripheral blood mononuclear cell of the cellsuspension.
 79. The method of claim 48, wherein said cell suspensioncomprises mammalian cells.
 80. The method of claim 79, wherein said cellsuspension comprises human, mouse, dog, cat, horse, monkey, or ratcells.
 81. The method of claim 48, wherein said compound comprises aliver cancer antigen.
 82. The method of claim 48, wherein said compoundcomprises a lung cancer antigen.
 83. The method of claim 48, whereinsaid compound comprises a bladder cancer antigen.
 84. The method ofclaim 48, wherein said compound comprises a breast cancer antigen. 85.The method of claim 48, wherein said compound comprises a colon cancerantigen.
 86. The method of claim 48, wherein said compound comprises arectal cancer antigen.
 87. The method of claim 48, wherein said compoundcomprises an endometrial cancer antigen.
 88. The method of claim 48,wherein said compound comprises a kidney cancer antigen.
 89. The methodof claim 48, wherein said compound comprises a leukemia antigen.
 90. Themethod of claim 48, wherein said compound comprises a melanoma antigen.91. The method of claim 48, wherein said compound comprises anon-Hodgkin lymphoma antigen.
 92. The method of claim 48, wherein saidcompound comprises a pancreatic cancer antigen.
 93. The method of claim48, wherein said compound comprises a prostate cancer antigen.
 94. Themethod of claim 48, wherein said compound comprises a thyroid cancerantigen.
 95. The method of claim 48, wherein said compound comprises anovarian cancer antigen.
 96. The method of claim 48, wherein saidcompound comprises a uterine cancer antigen.
 97. The method of claim 48,wherein said compound comprises an HIV antigen.
 98. The method of claim48, wherein said compound comprises an Ebola antigen.
 99. The method ofclaim 48, wherein said compound comprises an HPV antigen.
 100. Themethod of claim 48, wherein said compound comprises an EBV antigen. 101.The method of claim 64, wherein the compound comprises a protein orpeptide.
 102. The method of claim 78, wherein the peripheral bloodmononuclear cells comprise one or more of the following types of cells:T cells, B cells, NK cells, monocytes, dendritic cells, and macrophages.103. The method of claim 78, wherein the peripheral blood mononuclearcells comprise a T cell.
 104. The method of claim 78, wherein theperipheral blood mononuclear cells comprise a B cell.
 105. The method ofclaim 78, wherein the peripheral blood mononuclear cells comprise an NKcell.
 106. The method of claim 78, wherein the peripheral bloodmononuclear cells comprise a monocyte.
 107. The method of claim 78,wherein the peripheral blood mononuclear cells comprise a dendriticcell.
 108. The method of claim 78, wherein the peripheral bloodmononuclear cells comprise a macrophage.
 109. The method of claim 103,further comprising intracellular delivery of a compound that enhancesimmune cell function.
 110. The method of claim 103, further comprisingintracellular delivery of a compound that enhances T cell function. 111.The method of claim 110, wherein the compound that enhances T cellfunction is an immune checkpoint pathway inhibitor.
 112. The method ofclaim 103, further comprising intracellular delivery of a tolerogenicfactor.
 113. The method of claim 103, further comprising intracellulardelivery of an adjuvant.
 114. A method for engineering of immune cellfunction in an immune cell comprising intracellular delivery of acompound by passing a suspension comprising said immune cell through amicrofluidic device comprising a constriction and contacting the immunecell with said compound to deliver said compound into the cytosol ofsaid immune cell, wherein the constriction has a diameter of 2 μm to 10μm, wherein the compound comprises a protein comprising a TALEN protein,Zinc finger nuclease, or transcription factor.
 115. The method of claim114, wherein the immune cell displays a class I or class IIhistocompatibility antigen restricted processed form of an antigen on asurface of said immune cell.
 116. The method of claim 114, wherein saidmembrane is disrupted by passing said immune cell through a constrictionhaving a diameter of 3 μm to 6 μm.
 117. The method of claim 114, whereinsaid immune cell is a T cell, B cell, dendritic cell, macrophage, NKcell, monocyte, neutrophil, granulocyte, or innate lymphoid cell. 118.The method of claim 114, wherein said compound confers the expression ofa homing phenotype to the immune cell.
 119. The method of claim 114,wherein said compound induces the differentiation of the immune cellinto a cell with a tolerogenic phenotype.
 120. The method of claim 114,further comprising contacting said immune cell with an effector T celland activating a cytotoxic T cell immune response.
 121. The method ofclaim 114, further comprising contacting said immune cell with aneffector T cell and activating a helper T cell immune response.
 122. Themethod of claim 114, further comprising contacting said immune cell withan effector T cell and activating a tolerogenic T cell immune response.123. The method of claim 114, wherein said compound enhances immune cellfunction.
 124. The method of claim 123, wherein the immune cell is a Tcell.
 125. The method of claim 124, wherein the compound that enhances Tcell function is an immune checkpoint pathway inhibitor.
 126. The methodof claim 114, wherein said compound comprises a tolerogenic factor. 127.The method of claim 114, wherein the compound comprises a peptide. 128.The method of claim 114, wherein said immune cell is in a resting state.129. The method of claim 128, wherein expression of one or more of themarkers CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I,MHC-II, CD62L, CCR7, CX3CR1 or CXCR5 in the immune cell is modulated bydelivery of said compound into said immune cell.
 130. The method ofclaim 129, wherein expression of one or more of the markers is decreasedand/or expression of one or more of the markers is increased.
 131. Themethod of claim 114, wherein said immune cell is a naive immune cell ora memory immune cell.
 132. The method of claim 114, wherein said cellsuspension comprises whole blood, buffy coat cells, a mixed cellpopulation, or a purified cell population.
 133. The method of claim 114,wherein said cell suspension comprises peripheral blood mononuclearcells, and wherein the compound is delivered to a peripheral bloodmononuclear cell of the cell suspension.
 134. The method of claim 114,wherein said cell suspension comprises mammalian cells.
 135. The methodof claim 134, wherein said cell suspension comprises human, mouse, dog,cat, horse, monkey, or rat cells.
 136. The method of claim 114, whereinthe compound comprises a protein comprising a TALEN protein.
 137. Themethod of claim 114, wherein the compound comprises a protein comprisinga Zinc finger nuclease.
 138. The method of claim 114, wherein thecompound comprises a protein comprising a transcription factor.
 139. Themethod of claim 133, wherein the peripheral blood mononuclear cellscomprise one or more of the following types of cells: T cells, B cells,NK cells, monocytes, dendritic cells, and macrophages.
 140. The methodof claim 133, wherein the peripheral blood mononuclear cells comprise aT cell.
 141. The method of claim 133, wherein the peripheral bloodmononuclear cells comprise a B cell.
 142. The method of claim 133,wherein the peripheral blood mononuclear cells comprise an NK cell. 143.The method of claim 133, wherein the peripheral blood mononuclear cellscomprise a monocyte.
 144. The method of claim 133, wherein theperipheral blood mononuclear cells comprise a dendritic cell.
 145. Themethod of claim 133, wherein the peripheral blood mononuclear cellscomprise a macrophage.